CN113045626A - Polypeptide for inhibiting amyloid-beta focusing and application thereof - Google Patents

Polypeptide for inhibiting amyloid-beta focusing and application thereof Download PDF

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CN113045626A
CN113045626A CN202110362946.2A CN202110362946A CN113045626A CN 113045626 A CN113045626 A CN 113045626A CN 202110362946 A CN202110362946 A CN 202110362946A CN 113045626 A CN113045626 A CN 113045626A
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polypeptide
beta
amyloid
protein
polypeptide sequence
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CN113045626B (en
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张改平
王方雨
邢广旭
金前跃
孙雪峰
魏啬
曹帅
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Key Laboratory Of Animal Immunology Henan Academy Of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention relates to a polypeptide for inhibiting amyloid-beta focusing and application thereof, wherein a polypeptide ligand P2696 which has the best binding mode and affinity with a target protein in a virtual polypeptide database is searched by a molecular docking virtual screening technology on the basis of an amyloid-beta crystal structure by means of computer-aided design, and the polypeptide sequence is YEKKGM. Solid phase Synthesis of YEKKGM Using Abeta1‑42The results of experiments of thioflavin T ThT fluorescence, surface plasmon resonance and cytotoxicity on the protein standard respectively show that the polypeptide P2696 is used for A beta1‑42The protein aggregation has good binding capacity, thereby proving that the polypeptide designed by the invention can be used for inhibiting A beta1‑42Toxicity of the aggregates.

Description

Polypeptide for inhibiting amyloid-beta focusing and application thereof
Technical Field
The invention relates to a polypeptide for inhibiting amyloid-beta focusing and application thereof, belonging to the field of polypeptide design and drug screening and development.
Background
With the continuous development of science and technology in recent years, computer application is becoming more important in the field of biology, and the molecular docking virtual screening technology based on computer simulation is a research hotspot for rational design and screening of affinity peptides in recent years. The method is characterized in that continuous butt joint of polypeptide small molecules on active sites of target protein molecules is realized by means of computer fast operation, the polypeptides are derived from a prepared virtual peptide library, then small molecule polypeptides capable of being combined with the target proteins are found through virtual butt joint with the target proteins, the combination mode of the polypeptides and the target proteins is calculated through computer software and scored, ligands better combined with the target proteins are selected according to scoring results, and in-vitro experiment screening and verification are performed after synthesis.
The number of patients suffering from Alzheimer Disease (AD) in China exceeds 900 million, patients suffering from early mild cognitive impairment exceed 2300 million, the number of patients suffering from AD exceeds 2000 million by 2050, the people are countries with the largest AD population and the highest growth speed in the world, and heavy burden is brought to the patients, families, society and medical treatment. In the elderly population over 60 years of age, the risk of AD increases by approximately 1.85-fold for every 5 years of age. Therefore, the economic burden and social problems caused by AD are becoming more serious and have become one of the great challenges facing the whole human being. Excessive aggregation of the amyloid-beta structure (β -amyloid) and hyperphosphorylation of Tau protein are two major hypotheses that we consider the development of AD. Many scholars consider senile plaques formed by deposition of a β formed after cleavage of APP to be a major cause of the onset of AD, and therefore, prevention of a β aggregation is a promising approach to treat AD. The existing research proves that the soluble Abeta oligomer has the greatest toxicity, and mainly activates the approaches of neuroglia cell triggering inflammatory reaction and the like by influencing cell membrane ion channels, generating oxidative stress, and activating the neuroglia cell. A β exists in various forms in the human body, mainlyWith A beta1-42And Abeta1-40Mainly, wherein A beta1-42This has been the focus of research because it is more toxic and more prone to aggregation.
Disclosure of Invention
The invention searches a polypeptide ligand P2696 with the best binding mode and affinity with a target protein in a virtual polypeptide database by a molecular docking virtual screening technology on the basis of an amyloid-beta crystal structure by means of computer-aided design, wherein the polypeptide sequence is YEKKGM. Solid phase Synthesis of YEKKGM Using Abeta1-42The results of experiments of thioflavin T (ThT) ThT fluorescence, Surface Plasmon Resonance (SPR) and cytotoxicity on protein samples respectively show that the polypeptide P2696 is used for treating A beta1-42The protein aggregation has good binding capacity, thereby proving that the polypeptide designed by the invention can be used for inhibiting A beta1-42Toxicity of the aggregates.
In order to achieve the purpose, the invention adopts the technical scheme that:
a polypeptide sequence P2696 for inhibiting amyloid-beta focusing, wherein the polypeptide sequence P2696 is YEKKGM.
The polypeptide sequence P2696 comprises corresponding modifications of the polypeptide sequence P2696 by taking the polypeptide sequence P2696 as a core; the modified material comprises nano material, fluorescent material, enzyme and biotin.
The polypeptide sequence P2696 is applied to inhibiting the toxicity of the amyloid-beta structure.
The polypeptide sequence P2696 is applied to detecting an amyloid-beta structure.
The polypeptide sequence P2696 is applied to the preparation of drugs with amyloid-beta structures.
The invention has the beneficial effects that:
1. the invention obtains a polypeptide sequence P2696 specifically combined with beta-amyloid by a molecular docking virtual screening technology based on an amyloid-beta crystal structure (PDB ID:6SHS), wherein the polypeptide sequence is YEKKGM. Solid phase synthesizing polypeptide, performing affinity identification with amyloid-beta, and identifying P2696 sequence and A beta1-42Interaction between proteinsEquilibrium dissociation constant KDIs 2.693X 10-6M, indicates that the affinity is better.
2. The P2696 polypeptide sequence of the invention has no toxicity to PC12 cells to a certain extent and has no toxicity to A beta1-42The PC12 cell injury caused by the protein has better protective effect. The polypeptide designed and synthesized by the invention has the advantages of convenience, high efficiency and low cost.
Drawings
FIG. 1 shows the P2696 sequence and A.beta.1-42And (4) displaying the docking result of the protein.
FIG. 2 shows that the P2696 sequence inhibits A.beta.1-42Graph of the effect of protein aggregation.
Wherein, Abeta represents that only A beta is contained1-42A sample; p812696 denotes P2696 and A beta1-42The mixed sample of (1).
FIG. 3 shows the P2696 sequence and A.beta.1-42And (3) identifying the LSPR affinity of the protein.
Wherein the ordinate represents the signal value detected by the sensor; the abscissa represents the time of interaction of the sample in the sensor.
FIG. 4 shows the effect of the P2696 sequence itself on the viability of PC12 cells.
Wherein the abscissa represents the polypeptide concentration and the ordinate represents the cell viability.
FIG. 5 shows P2696 sequence and A.beta.at different concentrations1-42Protein co-incubation followed by A β -pairing1-42Effects of protein toxicity.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 molecular docking and screening of virtual peptide libraries
1、Aβ1-42Preparation of protein Crystal Structure
For Abeta by means of computer aided design software1-42The crystal structure of the protein (PDB ID:6SHS) was analyzed, and the 16 th to 23 th amino acid residues were selected as the designated docking region for molecular docking.
2. Design of virtual polypeptide libraries
The invention adopts a mode of prolonging amino acid residues one by one, firstly, several amino acid libraries with the highest scores are butted with the structure of a target protein one by one, the optimal amino acid residue is selected as a core according to the butting condition, and then the corresponding amino acid number is sequentially increased until the optimal butting result is reached. The peptide sequence generated by the virtual peptide library preferably has 3-12 amino acid residues.
3. Assessment of docking results
Respectively calculating the mechanical parameters of free energy, hydrogen chain, Van der Waals force, etc. of polypeptide and protein combination to make comprehensive evaluation so as to judge screening result, and screening to obtain P2696 whose polypeptide sequence is YEKKGM and Abeta1-42The results of the protein docking interactions are shown in FIG. 1.
Example 2P 2696 sequence and A β1-42Fluorescent intensity identification of protein binding
1. First is Abeta1-42Preparation of monomers from Abeta1-42Dissolving in Hexafluoroisopropanol (HFIP) at 1mg/mL, standing for 1 hr, ultrasonic treating for 10 min after dissolving sufficiently, volatilizing HFIP completely in a fume hood, storing in a refrigerator at-80 deg.C, and dissolving in PBS buffer (containing 100mM PB and 10mM NaCl, pH 7.4) when in use.
2. The obtained P2696 sequence was dissolved in PBS buffer (containing 100mM PB, 10mM NaCl, pH 7.4). During the experiment, Abeta is added1-42Mixing the monomer solution and the P2696 solution, and mixing the A beta1-42The final concentration was 25. mu.M and the final concentration of P2696 was 50. mu.M.
3. The mixed samples were incubated together in an incubator at 37 ℃ for 24 hours, and 20. mu.L of the sample was taken. ThT fluorescence intensity was measured at an excitation wavelength of 440nm and an emission wavelength of 480nm after 20-fold dilution with ThT staining solution (containing 25. mu.M ThT, 25mM PB). Will contain only A beta1-42The fluorescence intensity of the sample was set to 100%, and normalization processing was performed (see fig. 2).
The results show that the P2696 sequence is specific to the synthesized A beta1-42The in vitro aggregation of the protein has good inhibition effect.
Example 3P 2696 sequence and A β1-42Affinity identification of proteins
1. Determination of appropriate pH as couplingAnd (4) conditions. In the reaction of Abeta1-42Before the protein is fixed on the CM5 chip, a proper buffer solution pH needs to be screened, so that the ligand is enriched to the vicinity of the surface of the CM5 chip through electrostatic adsorption, and a better coupling effect is achieved. Separately, the A beta solution was diluted with sodium acetate solutions of pH 5.5, 5.0, 4.5, and 4.01-42The sample was 50. mu.g/mL. The loading time was 180s, 50mM NaOH was used as a washing solution, and the pH4.5 was used as a coupling condition according to the results.
2. And (3) ligand coupling. Immobilization of Abeta Using amino direct coupling1-42Proteins were applied to the surface of CM5 chips. Flow Cell 1 was selected as the reference channel and Flow Cell 2 as the sample channel. The coupling mode was chosen as specific contact time. Then according to the sample position diagram, HBS-EP buffer solution, EDC/NHS solution and A beta dissolved in sodium acetate with pH4.5 are correspondingly put in one to one1-42Adding the solution and ethanolamine solution into a sample tray, checking a buffer solution, and storing a method and a result file; click Run, begin formal coupling.
3. And (4) measuring the affinity. Run Kinetics/Affinity assay click Kinetics/Affinity was chosen to set the relevant experimental parameters, Flow Cell 1 and 2 and chip type CM5 were chosen. Startup's solution is HBS-EP buffer, binding time 120s, dissociation time 120s, regeneration solution is 0.25% Sodium Dodecyl Sulfate (SDS), and stabilization time 30 s. Filling out the sample name as P2696, dissolving the sample in HBS-EP buffer solution, diluting to polypeptide solution with concentration of 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, 1.56 μ M and 0.78 μ M, setting a sample with repeated zero concentration and minimum concentration, placing the sample as required, checking the buffer solution, storing the file, and clicking Run to start the experiment. Ligand coupling and affinity determination were both accomplished using Biacore X100 Control Software.
4. And (6) data processing. After the experiment, the results were analyzed by Evaluation software, and the background signal of Flow Cell 1 was subtracted from Flow Cell 2 to obtain the experimental results. The fitting was performed using a 1:1binding fitting method (see fig. 3). The concentration of the solution decreases gradually from the top to the bottom of the graph.
The results show that as the concentration of P2696 increases, the amount of binding also increases and the response time increasesFast, P2696 sequence for synthetic A.beta.1-42The protein has better affinity binding, and the P2696 sequence is used for synthesizing A beta1-42The protein has better affinity combination, and the equilibrium dissociation constant K of the interaction between the protein and the proteinDIs 2.693X 10-6M。
Example 4 toxicity characterization of P2696 Polypeptides
1. Polypeptide sample preparation. The P2696 polypeptide was dissolved in DMEM complete medium (DMEM high-sugar medium containing 10% fetal bovine serum, containing 4.5g/L glucose, L-glutamine, sodium pyruvate) to prepare polypeptide solutions of different concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200. mu.M) for further experiments.
2. And (5) plating cells. Selecting PC12 cells in logarithmic phase of growth, washing twice with PBS, adding 1-2mL trypsin, digesting at 37 deg.C for 1min, tapping the side of the culture flask, observing the cell state under an inverted microscope, adding DMEM complete culture medium to stop digestion when the cell gap becomes large and round, gently blowing the cells down, transferring to a centrifuge tube, centrifuging at 1000r/min for 5 min, discarding the supernatant, adding new DMEM complete culture medium, counting by a cell counting plate, and diluting to 5 × 10 density4Cell suspension at 100. mu.L/well (5X 10)3One/hole) planking.
3. The CCK-8 method is used for detecting the cell viability. After the cells are attached to the wall, polypeptide solutions with different concentrations are added, each concentration is 5 multiple wells, the drug administration group is used as a control group, only the culture medium solution is added, and 100 mu L of culture medium and 10 mu L of CCK-8 solution are added, but the wells without the cells are used as a blank group. After incubation at 37 ℃ for 24h, 10. mu.L of CCK-8 solution was added to each well, incubation was continued in a cell incubator for 2 hours, and absorbance was measured at 450 nm. Cell viability ═ 100% (dose-blank)/(control-blank) (see figure 4).
The result shows that with the increase of the concentration of the polypeptide P2696, the P2696 generates certain toxicity to PC12 cells at higher concentration, the effect is better at low concentration, and the P2696 sequence has no toxicity to PC12 cells to a certain extent.
Example 5 inhibition of A β by P2696 Polypeptides1-42Identification of toxicity
1. And (4) sample preparation. beta-Abeta prepared at the previous stage1-42The monomer and P2696 polypeptide are respectively dissolved in DMEM complete medium to obtain A beta 1-4225 μ M, 50 μ M P2696 polypeptide sample, incubated for 72h at 37 ℃.
2. And (5) plating cells. Selecting PC12 cells in logarithmic phase of growth, washing twice with PBS, adding 1-2mL trypsin, digesting at 37 deg.C for 1min, tapping the side of the culture flask, observing the cell state under an inverted microscope, adding DMEM complete culture medium to stop digestion when the cell gap becomes large and round, gently blowing the cells down and transferring to a centrifuge tube, centrifuging at 1000r/min for 5 min, discarding the supernatant, adding new DMEM complete culture medium, counting by a cell counting plate, diluting to 5 × 10 density4Cell suspension at 100. mu.L/well (5X 10)3One/hole) planking.
3. The CCK-8 method is used for detecting the cell viability. Respectively adding the A beta-containing solution after the cells are attached to the wall1-42Monomeric samples, and A beta in the presence of P2696 polypeptide inhibitors1-42Monomer sample (A beta)1-42The mixing volume of the monomer and the P2696 polypeptide is shown in figure 5), each concentration is 5 multiple wells, and the drug administration group is formed; a control group was prepared by adding only the medium solution, wells to which 100. mu.L of the medium and 10. mu.L of the CCK-8 solution were added but no cells were added were used as a blank group, and after culturing at 37 ℃ for 24 hours, 10. mu.L of the CCK-8 solution was added to each well, and the culture was continued in a cell incubator for 2 hours, and the absorbance at 450nm was measured. Cell viability ═ 100% (dose-blank)/(control-blank) (see fig. 5, a25 indicated a β concentration of 25 μ M).
The results show that A beta 1-42 has a large influence on the cell viability of PC12, and the cell viability is increased after the co-incubation of P2696 and A beta 1-42, wherein the ratio of A beta: the highest is reached when the polypeptide is 1:4, and the P2696 sequence is opposite to A beta1-42The damage of the PC12 cells caused by the method has better protective effect.
Sequence listing
<110> animal immunology key laboratory of academy of agricultural sciences in Henan province
<120> polypeptide inhibiting amyloid-beta focusing and application thereof
<130> amyloid-beta
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 1
Tyr Glu Lys Lys Gly Met
1 5

Claims (5)

1. A polypeptide sequence P2696 that inhibits amyloid-beta focusing, wherein said polypeptide sequence P2696 is YEKKGM.
2. The polypeptide sequence P2696 of claim 1, which comprises modifications of the polypeptide sequence P2696 with respect to the core polypeptide sequence P2696; the modified material comprises nano material, fluorescent material, enzyme and biotin.
3. Use of the polypeptide sequence P2696 of claim 1 or 2 for inhibiting toxicity of amyloid-beta structure.
4. Use of the polypeptide sequence P2696 of claim 1 or 2 for detecting a amyloid-beta structure.
5. Use of the polypeptide sequence P2696 of claim 1 or 2 for the preparation of a targeted drug of amyloid-beta structure.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104277105A (en) * 2013-07-12 2015-01-14 国家纳米科学中心 Polypeptide inhibitor for inhibiting aggregation and toxicity of beta amyloid protein and application of polypeptide inhibitor
WO2017088711A1 (en) * 2015-11-25 2017-06-01 中国科学院过程工程研究所 Polypeptide binding to a plurality of amyloid monomers and aggregates, and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104277105A (en) * 2013-07-12 2015-01-14 国家纳米科学中心 Polypeptide inhibitor for inhibiting aggregation and toxicity of beta amyloid protein and application of polypeptide inhibitor
WO2017088711A1 (en) * 2015-11-25 2017-06-01 中国科学院过程工程研究所 Polypeptide binding to a plurality of amyloid monomers and aggregates, and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈紫晗等: "β-淀粉样蛋白清除障碍在阿尔兹海默症发病中的作用", 《生理科学进展》, vol. 50, no. 2, pages 149 - 152 *

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