A small molecule active peptide and a preparation method and application thereof

Abstract

The present application relates to a kind of small molecule active peptides, and its amino acid sequence is: LPAYPMP.Preparation method is: with alkaline protease enzymatic hydrolysis abalone viscera, enzyme is destroyed after enzymolysis, and the enzymatic hydrolysis liquid is obtained;Enzymatic hydrolysis liquid is treated by centrifugation, coarse filtration and ultrafiltration, and a plurality of polypeptides with molecular weight less than 1kDa are collected and freeze-dried into powder respectively;Take polypeptide freeze-dried powder and carry out sequence identification, obtain a plurality of polypeptide sequences, delete repeated sequences, and screen a plurality of non-repeated polypeptides;With protein HMGR as receptor, the obtained polypeptides are virtually screened by docking software, and the polypeptides with high docking score are screened out, and small molecule active peptide is obtained by solid-phase synthesis.The small molecule active peptide of the present application can effectively inhibit the accumulation of total cholesterol and total triglyceride in cells, has high inhibition rate on cholesterol micelle solubility, has the effect of lowering blood lipid, can be applied to the preparation of auxiliary blood lipid-lowering drugs, and has the advantages of high specificity and small toxic and side effects compared with traditional blood lipid-lowering drugs.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a small molecule bioactive peptide, its preparation method, and its application. Background Technology

[0002] Currently, statins are the most commonly used lipid-lowering drugs in clinical practice. Reliable studies have shown that statin use has two main side effects: elevated transaminase levels and the risk of muscle pain. Therefore, controlling blood lipids through diet is gaining popularity. However, current processing and utilization of abalone mainly focuses on canning, freezing, and fresh sales, with low utilization of deep processing methods and significant waste of byproducts.

[0003] Existing research on abalone bioactivity mainly focuses on the antioxidant activity of gonads, viscera, shell, and byproducts such as gills. Currently, research on developing naturally derived lipid-lowering peptides is gaining popularity, but reports on lipid-lowering peptides derived from abalone viscera are scarce. Summary of the Invention

[0004] The purpose of this invention is to provide a small molecule active peptide, its preparation method and application. It uses abalone by-products as raw materials to obtain polypeptides with lipid-lowering effects, providing a new idea for the high-value processing and utilization of abalone by-products, and also expanding resources for the development of natural lipid-lowering products.

[0005] To achieve the above objectives, this invention discloses a small molecule bioactive peptide with the amino acid sequence LPAYPMP.

[0006] The preparation method of the above-mentioned small molecule bioactive peptides includes the following steps:

[0007] S1. Raw Material Preparation

[0008] The viscera of abalone are selected as the raw material;

[0009] S2. Enzymatic hydrolysis

[0010] The viscera of abalone were hydrolyzed using alkaline protease. After hydrolysis, the enzyme was inactivated to obtain the hydrolysate.

[0011] S3. Filtering

[0012] The enzymatic hydrolysate was centrifuged, coarsely filtered, and ultrafiltered to collect multiple polypeptides with a molecular weight of less than 1 kDa, which were then freeze-dried into powder.

[0013] S4. Sequence Identification

[0014] The lyophilized peptide powder was subjected to sequence identification, and multiple peptide sequences were obtained. Repeated sequences were deleted, and multiple non-repeating peptides were obtained by screening.

[0015] S5. Virtual Filtering

[0016] Using the protein HMGR as the receptor, multiple peptides were virtually screened using docking software. The peptides with the highest docking scores were then synthesized in a solid phase to obtain the aforementioned small molecule bioactive peptides.

[0017] Preferably, in step S2, the amount of alkaline protease added is 8000-12000 U / g substrate, the pH value is 9.5-10.5, the ratio of substrate to ultrapure water is 1:40-60, the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 3-6h.

[0018] Preferably, in step S2, the amount of alkaline protease added is 10000 U / g substrate, the pH value is 10.0, the ratio of substrate to ultrapure water is 1:50, the enzymatic hydrolysis temperature is 55℃, and the enzymatic hydrolysis time is 4h.

[0019] Preferably, in step S3, the enzymatic hydrolysate is centrifuged using a plate centrifuge, coarsely filtered using a ceramic membrane, and the filtrate is ultrafiltered using a membrane with a molecular weight of <1 kDa to collect multiple polypeptides with a molecular weight of less than 1 kDa.

[0020] Preferably, in step S4, after the lyophilized peptide powder is desalted by a desalting column, mass spectrometry analysis is performed using a nano-spray ion source mass spectrometer to obtain the peptide sequence.

[0021] Preferably, in step S5, the HMGR protein molecule is hydrogenated and dehydrated using the docking software Discovery Studio to minimize energy. The polypeptide molecule is drawn as a small molecule using Discovery Studio Client as a ligand. Finally, the active pocket of the HMGR protein is determined, and virtual screening is performed under the conditions of pocket range 8 and box edge 4.

[0022] The present invention also discloses a protein-peptide complex in which the peptide and the protein are bound together, wherein the amino acid sequence of the peptide is LPAYPMP and the protein is HMGR protein.

[0023] Preferably, the crystal structure of the HMGR protein is 1HWK.

[0024] In addition, the present invention also discloses the application of the above-mentioned small molecule active peptides and proteins in the preparation of adjuvant lipid-lowering drugs.

[0025] The present invention has the following beneficial effects:

[0026] 1. The small molecule active peptides of this invention can effectively inhibit the accumulation of total cholesterol and total triglycerides in cells, have a high inhibition rate of cholesterol micelle solubility, and have lipid-lowering effects. They can be used to prepare adjuvant lipid-lowering drugs and have the advantages of high specificity and low toxicity and side effects compared with traditional lipid-lowering drugs.

[0027] The small molecule active peptides of this invention are prepared from abalone viscera, making use of waste and avoiding the waste of precious abalone meat. This provides a new idea for the high-value processing and utilization of abalone by-products and expands resources for the development of natural lipid-lowering products. Attached Figure Description

[0028] Figure 1 This is an HPLC chromatogram of a small molecule bioactive peptide.

[0029] Figure 2 This is the mass spectrum of a small molecule bioactive peptide.

[0030] Figure 3 The effect of different concentrations of LPAYPMP on the viability of Hep-G2 cells.

[0031] Figure 4 The effect of different concentrations of LPAYPMP on total cholesterol (TC) in Hep-G2 cells.

[0032] Figure 5 The effect of different concentrations of LPAYPMP on total triglycerides (TG) in Hep-G2 cells.

[0033] Figure 6 The inhibition rate of cholesterol micelle solubility of different concentrations of LPAYPMP is shown.

[0034] Figure 7 The results show the molecular docking between 1HWK and LPAYPMP.

[0035] Figure 8 for Figure 7 Enlarged schematic diagram of part A in the middle.

[0036] Figure 9 This is a graph showing the root mean square deviation (RMSD) of all atoms in a protein-ligand complex relative to its initial structure.

[0037] Figure 10 The RMSF value represents the change in the amino acid backbone atoms of the protein-ligand complex over time.

[0038] Figure 11 This is a statistical diagram of protein-ligand complex interactions (categorized by amino acid participation).

[0039] Figure 12 A statistical graph of the interaction forces between proteins and small molecules (categorized by time). Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0041] This invention discloses a small molecule bioactive peptide with the amino acid sequence LPAYPMP, as shown in SEQ ID NO:1. This small molecule bioactive peptide can be used in the preparation of adjuvant lipid-lowering drugs.

[0042] Based on the same inventive concept, this invention also discloses a protein-peptide complex in which the peptide and the protein are bound together, wherein the amino acid sequence of the peptide is LPAYPMP, the protein is HMGR protein, and the crystal structure of the HMGR protein is 1HWK.

[0043] The preparation method of the above-mentioned small molecule bioactive peptides includes the following steps:

[0044] S1. Raw Material Preparation

[0045] The viscera of abalone are selected as the raw material.

[0046] S2. Enzymatic hydrolysis

[0047] Alkaline protease was used to enzymatically hydrolyze the viscera of abalone. The alkaline protease dosage was 8000–12000 U / g substrate, the pH was 9.5–10.5, the substrate-to-ultrapure water ratio was 1:40–60, the hydrolysis temperature was 40–60℃, and the hydrolysis time was 3–6 h. As a preferred embodiment, the alkaline protease dosage was 10000 U / g substrate, the pH was 10.0, the substrate-to-ultrapure water ratio was 1:50, the hydrolysis temperature was 55℃, and the hydrolysis time was 4 h. After hydrolysis, the enzyme was inactivated to obtain the hydrolysate.

[0048] S3. Filtering

[0049] The enzymatic hydrolysate was centrifuged using a plate centrifuge. After centrifugation, it was coarsely filtered through a ceramic membrane. The filtrate was then ultrafiltered through a membrane with a molecular weight of <1 kDa to collect multiple peptides with a molecular weight of less than 1 kDa, which were then freeze-dried into powder.

[0050] S4. Sequence Identification

[0051] The lyophilized peptide powder was sequenced, yielding multiple peptide sequences. Repeated sequences were removed, and multiple non-repeating peptides were selected. Specifically, the lyophilized peptide powder was desalted using a C18 desalting column, followed by mass spectrometry analysis using a nanospray ionization source to obtain the peptide sequences. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with a tandem EASY-nanoLC 1200. A total of 1 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated using a gradient at a rate of 60 min, with the column flow rate controlled at 300 nL / min, the column temperature at 40 °C, and the electrospray voltage at 2 kV. The gradient started at 2% B phase, increased non-linearly to 35% at 47 min, increased to 100% within 1 min, and maintained for 12 min. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows:

[0052] MS: Scan range (m / z): 200-2000; resolution: 70000; AGC target: 3e6; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 17500; AGC target: 1e5; maximum injection time: 45ms; collision energy: 28; dynamic exclusion time: 30s.

[0053] S5. Virtual Filtering

[0054] Using the protein HMGR with a crystal structure of 1HWK as the receptor, the crystal structure (PDB ID: 1HWK) of 3-hydroxy-3-methylglutaryl-CoA reductase (natural) was downloaded from the RCSB protein database (http: / / www.rcsb.org). The HMGR protein molecule was hydrogenated and dehydrated using the docking software Discovery Studio to minimize energy usage. Small molecules were plotted using Discovery Studio Client as ligands. Finally, the active pocket of the HMGR protein was determined, and virtual screening was performed with a pocket range of 8 and a box edge of 4.

[0055] The results of the virtual screening were ranked using Grid Score scores. Based on the ranking and interaction, peptides with stronger binding interactions with the pocket (i.e., peptides with higher scores) were selected for solid-phase synthesis to obtain the desired small molecule active peptides.

[0056] The small molecule bioactive peptides of this invention were synthesized using a solid-phase method, and the purity and molecular weight of the synthesized peptides were identified using HPLC and LC / MS. Figure 1As shown, HPLC analysis confirmed that the purity of LPAYPMP reached 99.53%. Figure 2 As shown, the relative molecular mass of LPAYPMP was determined to be 832.9 by LC / MS. The physicochemical parameters of LPAYPMP were analyzed using ProtParam, as shown in Table 1.

[0057] Table 1 Physicochemical parameters of small molecule bioactive peptides

[0058]

[0059] The toxicity and efficacy of the above-mentioned small molecule bioactive peptides were determined as follows:

[0060] 1. Cytotoxicity assay

[0061] Hep-G2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin. When the cells reached 80-90% confluence (logarithmic growth phase), they were digested, gently pipetted to mix, centrifuged to collect the cells, and diluted to a final concentration of 1. 10 5 Cells / mL. Add 100 μL of cell suspension to a 96-well plate and incubate at 37°C for 14-16 h in a CO2 incubator. When the cells reach 80-90% confluence, incubate the cells with different concentrations (0, 50, 100, 200, 400 μM) of the peptide LPAYPMP for 24 h. Determine cell viability according to the CCK8 kit method.

[0062] Effects of different concentrations of LPAYPMP on Hep-G2 cell viability, such as Figure 3 As shown in the figure. The results indicate that treatment with low concentrations of LPAYPMP (50 μM to 400 μM) had no significant effect on the cell viability of Hep-G2 cells, suggesting that peptide treatment at a concentration of 400 μM was non-toxic to Hep-G2 cells.

[0063] 2. Determination of TC and TG content

[0064] When cells reach the logarithmic growth phase, digest the cells, gently pipette to mix, centrifuge to collect the cells, add culture medium, pipette to mix again, count the cells using a hemocytometer, and dilute the cell count to 4% with complete culture medium. 10 5Cells / mL. Add 250 μL of cell suspension to a 48-well plate and incubate at 37°C for 14-16 h in a CO2 incubator. When the cells reach 80-90% confluence, add different concentrations (0, 50, 100, 200, 400 μM) of peptides and free fatty acids (10% oleic acid and palmitic acid) and incubate for 24 h. A blank control group without added free fatty acids and peptides is also included. Wash cells with DPBS and then lyse them on ice for 45 min using 1.5% Triton-X100. Measure the TC and TG content of the cells using the Nanjing Jiancheng Triglyceride (TG) assay kit and the Total Cholesterol (TC / TCH) assay kit.

[0065] Prepare protein standards of different concentrations. Mix reagent A and reagent B in a 50:1 ratio (prepare fresh for immediate use). Each well contains 25 μL of sample and 200 μL of reaction solution. Incubate in the dark for 30 min before measuring the OD562 nm of the reaction wells.

[0066] Total cholesterol (TC) content (mmol / g prot) = (Sample A - Blank A) / (Standard A - Blank A) C standard (5.17) ÷ Cpr

[0067] Total triglyceride (TG) content (mmol / g prot) = (Sample A - Blank A) / (Standard A - Blank A) C standard (2.26) ÷ Cpr

[0068] The effects of different concentrations of LPAYPMP treatment on total cholesterol (TC) accumulation in Hep-G2 cells are as follows: Figure 4 As shown. By Figure 4 It can be seen that treatment with different concentrations of LPAYPMP significantly inhibited the accumulation of total cholesterol in Hep-G2 cells, and the effect was best when the treatment concentration was 400 μM.

[0069] The effects of different concentrations of LPAYPMP treatment on total triglyceride (TG) accumulation in Hep-G2 cells are as follows: Figure 5 As shown. By Figure 5 It can be seen that different concentrations of LPAYPMP treatment inhibited the accumulation of total triglycerides in Hep-G2 cells. The accumulation of triglycerides in cells of each group gradually decreased with the increase of treatment concentration, and each treatment concentration was significantly different from the model group 0 μM.

[0070] 3. Determination of the in vitro cholesterol micelle solubility inhibition rate

[0071] The inhibition rate of peptides on cholesterol micelle solubility was determined using the method of Nagaoka et al., with slight modifications. The micelle solution (1 mL) contained 10 mM sodium taurine, 0.4 mM cholesterol, 1 mM oleic acid, 132 mM NaCl, and 15 mM sodium phosphate (pH 7.4). The peptide powder was dissolved in PBS to a final concentration of 10 mM, and then mixed with the cholesterol micelle solution to achieve final peptide concentrations of 50, 100, 200, and 400 μM. The mixed samples were ultrasonically emulsified at 37°C for 30 min and incubated with shaking at 37°C for 24 h. After centrifugation at 10000 r for 20 min at room temperature, the supernatant was used to determine the cholesterol content using a Nanjing Jiancheng total cholesterol test kit. A blank experiment was also performed, using an equal volume of PBS instead of the peptide solution. Finally, the inhibition rate of the peptide solution on cholesterol micelle solubility was calculated. The calculation formula is as follows:

[0072] Total cholesterol (TC) content (mmol / L) = Standard (5.17)

[0073] Cholesterol micelle solubility inhibition rate (%) =

[0074] The effect of different concentrations of LPAYPMP on the inhibition rate of cholesterol micelle solubility is as follows: Figure 6 As shown. By Figure 6 It can be seen that the inhibition rate of peptide treatment on cholesterol micelle solubility is enhanced with increasing treatment concentration, and the effect is most obvious when the treatment concentration is 400 μM.

[0075] 4. Molecular docking verification

[0076] The X-ray crystal structure of 1 HWK was obtained from a protein database. The protonation state of the small molecule was set to pH = 7.4, and the compound was expanded into a 3D structure using Open Babel. A series of preparations were performed on the receptor protein and ligand using the AutoDock tool (ADT3). The docking box was generated using the AutoGrid program, followed by molecular docking using Autodock Vina (1.2.0). The optimal binding conformation was selected to analyze interactions. Finally, a protein-ligand interaction map was generated using PyMOL.

[0077] The key amino acids for the docking of the protein HMGR with LPAYPMP in the 1HWK crystal structure are shown in Table 2.

[0078] Table 21 Key amino acids for HWK-LPAYPMP docking

[0079]

[0080] like Figure 7, 8 As shown, the 1HWK protein is represented by a dark blue cartoon model, the ligands by cyan stick models, and their binding sites by magenta stick structures. Nonpolar hydrogen atoms are omitted. Hydrogen bonds, ionic interactions, and hydrophobic interactions are depicted as yellow, magenta, and green dashed lines, respectively.

[0081] The discovered interactions were analyzed and categorized based on their interactions. Multiple sets of interactions were found between receptor proteins and small molecules, such as the hydrogen bonds formed between 1HWK's THR558 and GLU559 and their ligands. Under the influence of these interactions, the binding energy of the protein-small molecule complex was -8.2 kcal / mol, exhibiting excellent overall performance.

[0082] 5. Molecular dynamics simulation

[0083] Desmond / Maestro noncommercial version 2022.1 was selected as the kinetic simulation software. The complex system was equipped with TIP3P using the TIP3P water model, and a 0.15M sodium chloride solution was added to equilibrate the system. After minimizing and relaxing the system, a 100 ns molecular dynamics simulation was performed in an isothermal and isobaric system at 300 K and 1 bar. Trajectory coordinates were recorded every 100 ps. Molecular dynamics analysis was performed using Desmond's simulation interaction plot.

[0084] like Figure 9 As shown, the protein exhibited significant fluctuations during the simulation, with its RMSD stabilizing at around 45 Å. The RMSD of the ligand also fluctuated considerably, eventually stabilizing at around 24 Å. These results indicate that the initial conformation of the small molecule bound to the protein is unstable, and that the small molecule moved away from the binding pocket during the simulation.

[0085] like Figure 10 As shown, although the RMSF of the protein itself is relatively small, the RMSF of the small molecule itself is very large, which also confirms that the initial conformation of the small molecule and the protein is not stable, and that the small molecule has moved away from the binding pocket during the simulation.

[0086] like Figure 11 , 12 As shown, during the simulation, although the initial conformation was unstable, a significant number of interactions were formed. The hydrogen bond frequency formed by ARG568 reached as high as 92%, while the hydrogen bond frequency formed by GLn766 and GLN770 exceeded 80%. In addition, multiple hydrophobic interactions and water bridges were formed to facilitate their binding. In summary, we can conclude that although the small molecule and protein exhibited significant fluctuations, a new binding conformation was formed through kinetic simulation, under which the two molecules possess a certain affinity.

[0087] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A small molecule bioactive peptide, characterized in that: Its amino acid sequence is: LPAYPMP.

2. The use of the small molecule active peptide according to claim 1 in the preparation of adjuvant lipid-lowering drugs.

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