CN113061163A

CN113061163A - Peptide ligand targeting beta-amyloid 1-42 and application thereof

Info

Publication number: CN113061163A
Application number: CN202110362980.XA
Authority: CN
Inventors: 李宁; 王方雨; 黄现青; 宋莲军; 张蓓; 程永霞; 沈玥; 乔明武
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-07-02
Anticipated expiration: 2041-04-02
Also published as: CN113061163B

Abstract

The invention relates to a peptide ligand of a target beta-amyloid 1-42 and application thereof, which searches a peptide ligand P21 with the best combination mode and affinity with a target protein in a virtual peptide ligand database by a molecule docking virtual screening technology on the basis of a beta-amyloid crystal structure by means of computer-aided design, wherein the sequence of the peptide ligand is YVRHLK. Solid phase Synthesis of YVRHLK Using A β_1‑42The results of experiments of thioflavin T ThT fluorescence, surface plasmon resonance and cytotoxicity on protein samples respectively show that the peptide ligand P21 is used for treating A beta_1‑42The protein aggregation has good binding capacity and consists ofThis demonstrates that peptide ligands designed by the present invention can be used to inhibit A.beta._1‑42Toxicity of the aggregates.

Description

Peptide ligand targeting beta-amyloid 1-42 and application thereof

Technical Field

The invention relates to a peptide ligand of a targeting beta-amyloid 1-42 and application thereof, belonging to the field of peptide ligand design and drug screening 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 peptide aglucon small molecules on active sites of target protein molecules is realized by means of computer fast operation, the peptide aglucons are mostly from a prepared virtual peptide library, then the peptide aglucons capable of being combined with the target protein are found through virtual butt joint with the target protein, the combination mode of the peptide aglucons and the target protein is calculated through computer software and scored, ligands which are well combined with the target protein are selected according to the scoring result, and in-vitro experiment screening and verification are used 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. The excessive aggregation of amyloid beta-protein (A β) and hyperphosphorylation of Tau protein are two major hypotheses that we consider the development of AD. Many scholars believe that APP appears after cleavageSenile plaques formed by deposition of a β are a major cause of the onset of AD, and thus, prevention of a β aggregation is a promising approach for the treatment of 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 beta exists in various forms in human body, mainly in A beta_1-42And Abeta_1-40Mainly, wherein A beta_1-42This has been the focus of research because it is more toxic and more prone to aggregation.

Disclosure of Invention

The invention searches a peptide ligand P21 with the best combination mode and affinity with a target protein in a virtual peptide ligand database by a molecular docking virtual screening technology on the basis of a beta amyloid protein crystal structure by means of computer-aided design, and the sequence of the peptide ligand P21 is YVRHLK. Solid phase Synthesis of YVRHLK Using A β_1-42The results of experiments of thioflavin T (ThT) ThT fluorescence, Surface Plasmon Resonance (SPR) and cytotoxicity on protein samples respectively show that the peptide ligand P21 is used for A beta_1-42Protein aggregation has good binding capacity, thus proving that the peptide ligand designed by the invention can be used for inhibiting A beta_1-42Toxicity of the aggregates.

In order to achieve the purpose, the invention adopts the technical scheme that:

a peptide ligand P21 targeting beta-amyloid 1-42, wherein the sequence of the peptide ligand P21 is YVRHLK.

The peptide ligand P21 comprises a corresponding modification of the peptide ligand P21 with the peptide ligand P21 as a core; the modified material comprises nano material, fluorescent material, enzyme and biotin.

The peptide ligand P21 is used for inhibiting the toxicity of beta-amyloid 1-42.

The peptide ligand P21 is applied to the detection of beta-amyloid 1-42.

The peptide ligand P21 is applied to the preparation of the beta-amyloid 1-42 targeted drug.

The invention has the beneficial effects that:

1. the invention obtains a peptide ligand sequence P21 specifically combined with beta-amyloid by a molecular docking virtual screening technology based on a beta-amyloid crystal structure (PDB ID:6SHS), wherein the peptide ligand sequence is YVRHLK. Solid phase synthesis of peptide ligand, affinity identification of beta-amyloid protein, P21 sequence and A beta_1-42Equilibrium dissociation constant K of interactions between proteins_DIs 5.385X 10^-5M, indicates that the affinity is better.

2. The P21 peptide ligand sequence of the invention has no toxicity to PC12 cells to a certain extent and has no toxicity to A beta_1-42The PC12 cell damage caused by the protein has better protection effect, and has the advantages of convenience, high efficiency and low cost.

Drawings

FIG. 1 shows the sequence of P21 and A.beta._1-42And (4) displaying the docking result of the protein.

FIG. 2 shows that the P21 sequence inhibits A.beta._1-42Graph of the effect of protein aggregation.

Wherein A.beta.represents a compound containing only A.beta._1-42A sample; p21+ Abeta indicates P21 and Abeta_1-42The mixed sample of (1).

FIG. 3 shows the sequence of P21 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 P21 sequence on the viability of PC12 cells.

Wherein the abscissa represents the peptide ligand concentration and the ordinate represents the cell viability.

FIG. 5 shows the P21 sequence and A.beta.at different concentrations_1-42Protein co-incubation followed by A β -pairing_1-42Effects 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、Aβ_1-42Preparation of protein Crystal Structure

For Abeta by means of computer aided design software_1-42The 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 peptide ligand 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 sequences generated from the library of virtual peptide ligands preferably have amino acid residues from 3 to 12.

3. Assessment of docking results

Respectively calculating the binding free energy of peptide ligand and protein, hydrogen chain and Van der Waals force, etc. to make comprehensive evaluation, and using Cscore value to judge screening result to obtain P21, its peptide ligand sequence is YVRHLK, and its peptide ligand sequence is connected with Abeta_1-42The results of the protein docking interactions are shown in FIG. 1.

Example 2P 21 sequence and A.beta._1-42Fluorescent intensity identification of protein binding

1. First is Abeta_1-42Preparation of monomers from Abeta_1-42Dissolving 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, pH7.4) when in use.

2. The obtained P21 sequence was dissolved in PBS buffer (containing 100mM PB, 10mM NaCl, pH 7.4). During the experiment, Abeta is added_1-42Mixing the monomer solution and the P21 solution, and mixing A beta_1-42The final concentration was 25. mu.M, and the final concentration of P21 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 beta_1-42The fluorescence intensity of the sample was set to 100%, and normalization treatment was performed (See fig. 2).

The results show that the P21 sequence is specific to the synthesized A beta_1-42The in vitro aggregation of the protein has good inhibition effect.

Example 3P 21 sequence and A β_1-42Affinity identification of proteins

1. The appropriate pH was determined as the coupling conditions. In the reaction of Abeta_1-42Before 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. Diluting A beta with sodium acetate solution of pH5.5, 5.0, 4.5, 4.0_1-42The sample was 50. mu.g/mL. The loading time was 180s, 50mM NaOH was used as a washing solution, and pH4.5 was used as a coupling condition according to the results.

2. And (3) ligand coupling. Immobilization of Abeta Using amino direct coupling_1-42Proteins 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 one_1-42Adding 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. Fill out sample name P21, dissolve the sample in HBS-EP buffer, dilute to peptide ligand solution with concentration of 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M, 0.78. mu.M, and set a zero concentration and a minimum concentration repeat sample, place the sample as required, check the buffer, save the file, click 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 P21 is increased, the binding amount is larger, the response time is fast, and the P21 sequence is applied to the synthesized A beta_1-42The protein has better affinity combination, and the equilibrium dissociation constant K of the interaction between the protein and the protein_DIs 5.385X 10^- ⁵M。

Example 4 toxicity characterization of P21 peptide ligands

1. Peptide ligand sample preparation. The P21 peptide ligand was dissolved in DMEM complete medium (DMEM high-glucose medium containing 10% fetal bovine serum, containing 4.5g/L glucose, L-glutamine, and sodium pyruvate) to prepare peptide ligand solutions of various concentrations (3.125, 6.25, 12.5, 25, 50, 100, and 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, peptide ligand solutions with different concentrations are added, each concentration is 5 multiple wells, the administration group is used as the administration group, the control group is added with only the culture medium solution, and the blank group is used as the well added with 100 mu L of the culture medium and 10 mu L of CCK-8 solution but not added with the cells. 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 results show that the toxicity of the P21 is increased with the increase of the concentration of the P21, but the P21 sequence has no statistical difference and is not toxic to PC12 cells to a certain extent.

Example 5P 21 peptide ligands inhibit A.beta._1-42Identification of toxicity

1. And (4) sample preparation. beta-Abeta prepared at the previous stage_1-42The monomer and the P21 peptide ligand are respectively dissolved in DMEM complete medium to obtain the final concentration of Abeta_1-42mu.M, 50. mu.M sample of P21 peptide ligand was incubated at 37 ℃ for 72 h.

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 Abeta after the cells adhere to the wall_1-42Monomer sample, and A beta in the presence of P21 peptide ligand inhibitor_1-42Monomer sample (A beta)_1-42Mixed volumes of monomer and P21 peptide ligand such as in fig. 5), 5 multiple wells per concentration, for administration groups; 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 figure 5).

The results show that the A beta 1-42 has a large influence on the cell viability of the PC12, and the cell viability is increased after the P21 and the A beta 1-42 are incubated together, and the cell viability is increased when the A beta: the highest is reached when the peptide ligand is 1:4, P21 sequence pair A beta_1-42The damage of the PC12 cells caused by the method has better protective effect.

Sequence listing

<110> Henan university of agriculture

<120> a peptide ligand targeting beta-amyloid 1-42 and application thereof

<130> amyloid beta 1-42

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Artificial sequence ()

<400> 1

Tyr Val Arg His Leu Lys

1 5

Claims

1. A peptide ligand P21 targeting beta-amyloid 1-42, wherein the sequence of the peptide ligand P21 is YVRHLK.

2. The peptide ligand P21, according to claim 1, wherein the peptide ligand P21 is the core and the peptide ligand P21 is modified accordingly; the modified material comprises nano material, fluorescent material, enzyme and biotin.

3. Use of the peptide ligand P21 of claim 1 or 2 for inhibiting the toxicity of β -amyloid 1-42.

4. Use of the peptide ligand P21 of claim 1 or 2 for detecting β -amyloid 1-42.

5. Use of the peptide ligand P21 of claim 1 or 2 for the preparation of a medicament for targeting β -amyloid 1-42.