Dipeptide compound combinations that inhibit aβ42 aggregation and reduce its cytotoxic effects

Abstract

β-amyloid 1-42 (Aβ42) is a pathological marker protein and pathogenic factor in Alzheimer's disease. This invention, through molecular docking, molecular dynamics calculations, and pharmacological experiments, has developed and disclosed eight dipeptide compounds and their compound mixtures that inhibit Aβ42 aggregation and reduce its cytotoxicity. These dipeptide compounds include histidine-arginine (HR), histidine-tryptophan (HW), arginine-phenylalanine (RF), arginine-arginine (RR), arginine-tryptophan (RW), arginine-tyrosine (RY), tryptophan-arginine (WR), and arginine-methionine (RM). These eight dipeptide compounds and their compound mixtures can affect Aβ42 aggregation and its cytotoxic effects in the pathological progression of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology drugs, specifically to a drug that inhibits β-amyloid protein 1-42 (Aβ), a characteristic protein of Alzheimer's disease. 42 It is a compound composed of dipeptide compounds that aggregate and reduce their cytotoxicity. Background Technology

[0002] Alzheimer's disease (AD), also known as senile dementia, is a neurodegenerative disease. Currently, there is no clear cause. Its early onset is insidious and easily goes unnoticed. Later, the disease worsens and progresses slowly. Clinical manifestations include cognitive and memory impairment, progressive decline in daily living abilities and behavioral disorders, ultimately leading to death. There are currently no effective prevention or treatment methods.

[0003] The care and treatment of Alzheimer's patients are extremely expensive, in addition to the anti-Aβ drugs approved by the FDA in recent years. 42 Aside from monoclonal antibodies, there are currently no drugs that can alter or slow the pathological progression of Alzheimer's disease; they can only alleviate patient suffering and improve some symptoms such as memory decline to a certain extent. Given this situation, there is an urgent need to develop new drugs that target the main pathological processes of Alzheimer's disease.

[0004] The main pathological feature of AD is the presence of Aβ in the brain. 42 Aggregates to form plaques. It is produced by the hydrolysis of amyloid precursor protein APP by β and γ secretases, of which Aβ... 42 Accumulation of Aβ can damage synapses and lead to cognitive decline; therefore, inhibiting Aβ... 42 Aggregated compounds may become potential drugs for the prevention and treatment of Alzheimer's disease (AD). According to our analysis, Aβ... 42 The aggregation process is a spontaneous process of decreasing the system's free energy, and includes the following steps: First, Aβ 42 First, the hydrophobic monomers aggregate into oligomers, becoming aggregation nuclei; second, the oligomers continuously extend into Aβ. 42 Fibers; third, fibers aggregate into fiber bundles; fourth, fiber bundles deposit to form patches. This invention utilizes our constructed dipeptide structure database consisting of 20 L-α-amino acids, with Aβ... 42 Using the five-membered oligomeric protein structure (aggregation core) as a target, a complex mixture consisting of eight dipeptide compounds (histidine-arginine (HR), histidine-tryptophan (HW), arginine-phenylalanine (RF), arginine-arginine (RR), arginine-tryptophan (RW), arginine-tyrosine (RY), tryptophan-arginine (WR), and arginine-methionine (RM)) was developed through molecular docking, molecular dynamics analysis, and molecular pharmacology experiments. This mixture can interact with the five-membered polymeric Aβ protein.42 Binding, thereby inhibiting Aβ 42 The aggregation process reduces Aβ. 42 Cytotoxicity. Compared to other substances, small molecule peptides interact with target proteins according to the principles of protein-protein interactions, easily cross the blood-brain barrier, and also have advantages such as good biocompatibility and low toxicity. Summary of the Invention

[0005] 1. Purpose of the invention

[0006] Develop a method that inhibits Aβ 42 Aggregation effect, reducing Aβ 42 Cytotoxic dipeptide compounds and their compound mixtures.

[0007] 2. Technical solution of the invention

[0008] 1. Use the Python language to write ChemScript scripts to create a structural database of all dipeptides composed of 20 L-α amino acids.

[0009] II. Five-membered oligomer Aβ was obtained by screening from the Protein Database (PDB). 42 The protein molecule structure (number 5oqv) is the target protein conformation.

[0010] III. Using the five-membered oligomer Aβ 42 Using the open-source software AutoDock and our pre-built dipeptide structure database as target proteins, we performed molecular docking screening to identify dipeptides with the optimal docking state.

[0011] IV. Analysis of the five-membered polymeric state Aβ using GROMACS molecular dynamics software. 42 (5oqv) Binding stability with 8 selected dipeptides (HR, HW, RF, RR, RW, RY, WR and RM) (measured by the root mean square deviation (RMSD) of heavy elements in the dipeptide molecular structure).

[0012] V. Using GROMACS molecular dynamics software, the docking of the eight selected dipeptides with the five-membered polymeric state Aβ was analyzed. 42 The interaction energy of (5oqv) consists of the Lanner-Jones potential and the electrostatic Coulomb potential.

[0013] VI. Pharmacological Experimental Analysis: Eight dipeptides and their compound mixtures inhibited Aβ. 42 Aggregate and reduce Aβ 42 The resulting cytotoxic effects.

[0014] 3. Effects of the invention

[0015] This invention is the first to utilize the Python language to construct a dipeptide database, and through molecular docking, it screens for dipeptides that can interact with Aβ. 42 Eight dipeptides bound at different sites were identified, and molecular dynamics analysis was used to analyze the interactions of these eight dipeptides (HR, HW, RF, RR, RW, RY, WR, and RM) with Aβ. 42 Compared to traditional research methods, studying protein-protein interactions significantly saves manpower and resources and improves work efficiency. Based on this, pharmacological experiments were designed and implemented to verify the effects of these eight dipeptide compounds and their compound mixtures on Aβ. 42 Aggregation has a good inhibitory effect, reducing the secretion of Aβ. 42 Cytotoxic effects on the SH-SY5Y neural cell line. Based on the pathogenesis of Alzheimer's disease (AD), a compound mixture of these eight dipeptide compounds, when used in drugs or foods, can alter Aβ levels. 42 Pathological processes such as aggregation and the resulting cytotoxic effects. Attached Figure Description

[0016] Figure 1 .Aβ 42 The process of accumulating and depositing in the brain.

[0017] Figure 2 Chemical structures of eight dipeptide compounds obtained by molecular docking. HR: Histidine-arginine; HW: Histidine-tryptophan (HW); RF: Arginine-phenylalanine; RR: Arginine-arginine; RW: Arginine-tryptophan; RY: Arginine-tyrosine; WR: Tryptophan-arginine; RM: Arginine-methionine.

[0018] Figure 3 The 8-dipeptide obtained by molecular docking and the pentagonal polymerized Aβ 42 A schematic diagram of the combination. The strip-like structure represents the pentagonal aggregate state Aβ. 42 Spatial conformations. HR: Histidine-arginine; HW: Histidine-tryptophan (HW); RF: Arginine-phenylalanine; RR: Arginine-arginine; RW: Arginine-tryptophan; RY: Arginine-tyrosine; WR: Tryptophan-arginine; RM: Arginine-methionine.

[0019] Figure 4 Molecular dynamics analysis of eight dipeptide compounds and their pentagonal polymeric state Aβ 42 The stability of the binding. RMSD: Root Mean Square Deviation, representing the degree of spatial variation of heavy elements in the dipeptide chemical structure. HR: Histidine-Arginine; HW: Histidine-Tryptophan (HW); RF: Arginine-Phenylalanine; RR: Arginine-Arginine; RW: Arginine-Tryptophan; RY: Arginine-Tyrosine; WR: Tryptophan-Arginine; RM: Arginine-Methionine.

[0020] Figure 5 Molecular dynamics analysis of eight dipeptide compounds and their pentagonal polymeric state Aβ 42 The interaction energy is the sum of the Lanner-Jones potential energy and the electrostatic Coulomb potential energy. A more negative value indicates a stronger attraction between the two groups. HR: Histidine-Arginine; HW: Histidine-Tryptophan (HW); RF: Arginine-Phenylalanine; RR: Arginine-Arginine; RW: Arginine-Tryptophan; RY: Arginine-Tyrosine; WR: Tryptophan-Arginine; RM: Arginine-Methionine.

[0021] Figure 6 Pharmacological experiments analyzed eight dipeptide compounds and their compound mixtures to inhibit Aβ. 42 Aggregate and reduce Aβ 42 Cytotoxic effects. A: Eight dipeptide compounds and their mixtures inhibit Aβ. 42 The effect of aggregation. Aβ control group: only Aβ 42 The control group, which did not contain any dipeptide compounds, and the HR, HW, RF, RR, RW, RY, WR, RM, or combination groups were experimental groups that, in addition to the Aβ control group, included different dipeptide compounds or a combination mixture containing the above eight dipeptides. B: The eight dipeptide compounds and their combination mixtures reduced Aβ. 42 The cytotoxic effect caused. Aβ control group: only secreted Aβ. 42 The control group did not contain any dipeptide compounds. The HR, HW, RF, RR, RW, RY, WR, RM, or combination groups were experimental groups that, in addition to the Aβ control group, included different dipeptide compounds or a mixture of the above eight dipeptides. HR: Histidine-arginine; HW: Histidine-tryptophan (HW); RF: Arginine-phenylalanine; RR: Arginine-arginine; RW: Arginine-tryptophan; RY: Arginine-tyrosine; WR: Tryptophan-arginine; RM: Arginine-methionine. *P<0.05, **P<0.01, ***P<0.001. Statistical analysis compared with the Aβ control group was performed using one-way ANOVA, and Fisher's test was used to analyze the statistical significance of the differences between the two groups. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, and will be carried out in the following steps:

[0023] Specific Implementation Method 1: Analyzing dipeptide compounds and pentagonal polymeric Aβ using information science and technology. 42 The method of interaction.

[0024] Step 1: Construction of the dipeptide compound structure database. A ChemScript script was written in Python to create a database in sdf (Structure DataFile) format containing the structures of all dipeptide compounds, composed of random combinations of 20 L-α-amino acids.

[0025] Step 2: Search the Protein Data Bank (PDB) of the Research Consortium for Structural Bioinformatics (RCSB) and download the polymerized Aβ. 42 The three-dimensional structural data (number 5oqv) is available in the downloaded structural file. The downloaded file contains ternary and pentary aggregate states of Aβ. 42 Structure. Delete the ternary coincident state structure data and retain the 5-ary aggregate state Aβ. 42 The structural data was renamed to 5oqvPentamer.pdb and saved as the target protein structure file for molecular docking.

[0026] Step 3: Using the open-source molecular docking software AutoDock, with 5oqvPentamer as the docking acceptor, molecular docking was performed using the dipeptide compound structure database constructed in Step 1. The docking results were sorted according to the optimized binding energy, and the eight dipeptide compounds with the best docking states were selected for subsequent molecular dynamics and pharmacological analysis.

[0027] The eight dipeptide compounds identified through molecular docking screening are HR, HW, RF, RR, RW, RY, WR, and RM. Their chemical structures are shown in the attached figure. Figure 2 As shown.

[0028] Step 4: Molecular dynamics analysis of 8 dipeptide compounds and Aβ 42 The stability and interaction energy of the binding were analyzed. GROMACS molecular dynamics analysis software (version 2020.03) was installed on an Ubuntu (version 18.04) Linux operating system, employing a GPU hybrid parallel computing mode supported by CUDA (Compute Unified Device Architecture). Utilizing the AMBER99SB force field optimized for ab initio protein structure calculations and the TIP3P explicit water molecule model, the interaction energy of eight dipeptide compounds with Aβ was analyzed. 42 The stability and interaction energy of the combination. The system unit is defined as a periodic boundary and Aβ. 42 The closest distance is 3.5 nm to a regular dodecahedral box. Water molecules are filled into the system unit, and then Na... + and Cl -Randomly replacing water molecules makes the system electrically neutral. After minimizing the system energy and achieving equilibrium at temperature and pressure, molecular dynamics analysis is performed to calculate the relationship with Aβ. 42 The root mean square deviation (RMSD) of the positions of heavy elements other than hydrogen atoms in the combined dipeptide molecule structure. Figure 4 ); and Aβ 42 Lanner-Jones potential and Coulomb potential (electrostatic force) between the dipeptide molecule and the Aβ. The RMSD value of the heavy element position variation in the dipeptide molecule reflects the relationship between the dipeptide molecule and Aβ. 42 The stability of the combination; the sum of the Lanner-Jones potential and the Coulomb potential is the interaction energy ( Figure 5 The more negative the value, the stronger the gravitational pull between them.

[0029] The analysis results show that HW and RR are related to Aβ 42 The binding with β is the most stable. The other six dipeptides bind to Aβ. 42 The combined RMSD value can remain stable even after a short period of time. Figure 5 ).

[0030] Specific Implementation Method Two: Utilizing ThT to detect the inhibition of Aβ by dipeptide compounds and their compound mixtures. 42 The effect of aggregation. Aβ 42 By assembling in a β-sheet conformation, three hydrophobic regions are created, burying hydrophobic amino acids inside the protein complex, thereby lowering the system's free energy. ThT is a commonly used protein for aggregated Aβ states. 42 The benzothiazole fluorescent dye can specifically bind to Aβ. 42 The β-sheet structure of the ThT protein enhances fluorescence upon binding. Aβ in the system... 42 The more polymeric β-sheet structures there are, the stronger the fluorescence intensity of the system. ThT binds to Aβ 42 The outer side of the polymerized fiber does not interfere with Aβ. 42 Therefore, ThT is often used to analyze and determine Aβ. 42 The formation of polymeric fibers.

[0031] Step 1, Aβ 42 And dipeptide sample processing. Aβ 42 The powder was dissolved in 1.0% NH4OH to prepare a 1 mM stock solution. The dipeptide compound solid powder was dissolved in 10 mM phosphate-buffered saline (pH 7.4) (PBS) to prepare a 1 mM stock solution. In subsequent experiments, all stock solutions were diluted with 10 mM PBS to prepare working solutions of different concentrations just before use.

[0032] Step 2: Using thiosulfate T (ThT), fluorescence detection of dipeptide compounds and their mixtures on Aβ. 42Inhibition of aggregation. 0.0128 mg of ThT powder was dissolved in PBS, stirred thoroughly until dissolved, and then brought to a final volume of 10 mL to prepare a 4 mM ThT stock solution. In a 96-well plate with a black-walled, transparent bottom, Aβ was added sequentially to a final concentration of 10 μM. 42 8 μM of a dipeptide compound or a mixture of dipeptides (1 μM of each dipeptide) and 16 μM of ThT were used in a total reaction volume of 125 μL, with 5 replicates per group. After thorough mixing, the samples were incubated at 37°C for 24 hours. After incubation, the samples were placed in a Synergy H1 microplate reader, with an excitation wavelength of 450 nm, and fluorescence intensity was detected at a wavelength of 485 nm. The results are as follows. Figure 6 As shown in Figure A.

[0033] Specific Implementation Method 3: Using MTT compounds, we analyzed the effect of dipeptide compounds and their compound mixtures on reducing the Aβ secretion expression of the human neuroblastoma cell line SH-SY5Y. 42 The cytotoxic effect. The SH-SY5Y cell line, after being transfected with cells expressing Aβ, exhibited cytotoxic activity. 42 Four hours after inserting the pcDNA3.1 plasmid (with a secretion signal peptide added to the N-terminus), cells were counted at a rate of 5 x 10-1. 3 The sample was divided into each well of a 96-well plate. For the experimental group, a final concentration of 8 μM of the dipeptide compound or its compound mixture (1 μM of each dipeptide) was added to the untransfected Aβ plate. 42 Cells expressing the plasmid served as the blank control group, while cells without the dipeptide compound served as the Aβ control group. After culturing at 37°C in a 5% CO2 incubator for 48 hours, the culture medium was aspirated, and 90 μL of serum-free culture medium and 10 μL of MTT solution (5 mg / mL) were added to each well. After 4 hours, the solution in each well was aspirated, and 100 μL of DMSO was added. The cells were shaken at 37°C for 10 minutes. The absorbance of each well was measured at 570 nm using a Synergy H1 microplate reader. The cell viability of each well was calculated using the average value of the blank control group as 100% of the baseline. Figure 6 B).

Claims

1. The application of arginine-arginine dipeptide compound (RR) in the preparation of an inhibitor of aggregation of Aβ42, a characteristic protein of Alzheimer's disease, wherein the inhibitor is used solely for the treatment of Alzheimer's disease.

2. Application of arginine-arginine dipeptide compound (RR) in the preparation of a cytotoxic inhibitor of Aβ42, a characteristic protein of Alzheimer's disease, wherein the inhibitor is for the treatment of Alzheimer's disease only.

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