Hypolipidemic polypeptide as well as screening method and application thereof

Abstract

The invention provides a blood fat reducing polypeptide as well as a screening method and application thereof, and belongs to the field of polypeptides. The blood fat reducing polypeptide provided by the invention comprises at least one of 116 polypeptides with amino acid sequences of FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY and the like. The blood fat reducing polypeptide provided by the invention can effectively reduce blood fat, has no obvious toxic or side effect, shows good inhibitory activity on steroid esterase and lipase in an in-vitro enzyme activity test, and also can effectively reduce blood fat in an animal in-vivo experiment; and no obvious toxic or side effect is shown in toxicity prediction and animal in-vivo models.

Description

Technical Field

[0001] The present application relates to the field of polypeptides, and specifically to a lipid-lowering polypeptide and a screening method and application thereof. Background Art

[0002] Hyperlipidemia is one of the most common diseases in the world, which can cause a series of related diseases such as cardiovascular disease. In 2030, the number of deaths from cardiovascular diseases worldwide is expected to reach 22.3 million. The prevention and treatment of hyperlipidemia is of great significance.

[0003] There are many types of lipid-lowering drugs. Although a large amount of evidence-based medical evidence shows that statins are the cornerstone of preventing and treating atherosclerotic cardiovascular diseases, clinical data show that about 30% of patients still cannot achieve ideal blood lipids after long-term use of statins. On the one hand, some patients find it difficult to take statins in sufficient doses due to side effects of statins (such as myalgia and rhabdomyolysis); on the other hand, intensive dose statin therapy increases the incidence of new-onset diabetes, further limiting the use of the maximum dose of statins.

[0004] Peptide drugs have unique advantages and clinical application value in drug development due to their high biological activity and strong specificity. The products of peptide degradation are amino acids, which generally do not accumulate in specific organs and tissues and are easily cleared from the body through the liver and kidneys, so there are almost no side effects. Compared with large molecule biological drugs, peptides have lower immunogenicity, higher stability and lower commercial costs.

[0005] Therefore, it is necessary to develop peptide drugs that can effectively inhibit hypolipidemic effects and have lower toxic side effects. Summary of the invention

[0006] In view of this, the present application provides a lipid-lowering polypeptide and a screening method and application thereof, aiming to find peptide drugs that effectively inhibit lipid-lowering and have lower toxic side effects.

[0007] The embodiment of the present application is implemented by providing a lipid-lowering polypeptide. The lipid-lowering polypeptide includes one or more of the 116 polypeptides with the following amino acid sequences:

[0008] FPFFF, FFPFF, FFFFFP, PFFFF, FFFPFF, FFFPFW, FFFPFL, FFFPFG, FFFPFY, FFFPFI, FFFPFA, FFFPFV, FFFF PF, WFFFPF, LFFFPF, GFFFPF, YFFFPF, IFFFPF, AFFFPF, VFFFPF, FFFF PFF, FFFF PF W, FFFF PFL, FFFF PFG, FFFF PFY, FFFF PFI, FFFF PFA, FFFF PFV, FFFFFPF, FWFFFPF, FLFFFPF, FGFFFPF, FYFFFPF, FIFFFPF, FAFFFPF, FVFFFPF, FFFPFFF, FFFPFWF, FFFPFLF, FFFPFGF, FFFPFYF, FFFP FIF, FFFP AFF, FFFP FVF, FFFF PFF, WFFFPFF, LFFFPFF, GFFFPFF, YFFFPFF, IFFFPFF, AFFFPFF, VFFFPFF, FFFP FFW, FFFP FWW, FFFP FLW, FFFP FGW, FFFP FYW, FFFP FIW, FFFP FAW, FFFP FVW, FFFF PF W, WFFFPFW, LFFFPFW, GFFFPFW, YFFFPFW, IFFFPFW, AFFFPFW, VFFFPFW, WFFFPFF, WFFFPFW, WFFFPFL, WFFFPFG, WFFFPFY, WFFFPFI, WFFFPFA, WFFFPFV, WFFFFPF, WWFFFPF, WLFFFPF, WGFFFPF, WYFFFPF, WIFFFPF, WAFFFPF, WVFFFPF, WFFFPFL, FFFPFFL, FFFPFWL, FFFFFPFL, LWFFFPF, LFFFPFF, LFFFPFW, LFFFFPF, GFFFPFL, AFFFPFL, FFFP FLL, YFFFPFL, LGFFFPF, LFFFPFL, LFFFPFL, LYFFFPF, FFFP FGL, FFFP FYL, IFFFPFL, LAFFFPF, FFFP FIL, VFFFPFL, LIFFFPF, LFFFPFG, LLFFFPF, LFFFPFY, FFFP FAL, FFFP FVL, LFFFPFI, LVFFFPF, LFFFPFA, LFFFPFV.

[0009] In some embodiments, the hypolipidemic polypeptide comprises at least one polypeptide having an amino acid sequence of FFPFF, VFFFPF, PFFFF, AFFFPFF, or LFFFPFY.

[0010] In some embodiments, the amino acid sequence of the lipid-lowering polypeptide is FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY.

[0011] In some embodiments, the lipid-lowering polypeptide comprises PFFFF; or, the amino acid sequence of the lipid-lowering polypeptide is PFFFF.

[0012] The present application also provides a method for screening lipid-lowering polypeptides, comprising:

[0013] Provide the peptides to be screened, predict the probability of biological activity through the Peptide Ranker database, and screen out peptides with a biological activity prediction score > 0.8;

[0014] The peptides with biological activity prediction scores>0.8 were screened out by molecular docking to obtain lipid-lowering peptides.

[0015] In some embodiments, the peptide segment to be screened is a polypeptide having 5-7 amino acids; and / or,

[0016] The proportion of hydrophobic amino acids in the peptide to be screened is ≥80%.

[0017] In some embodiments, the screening of the peptides with predicted biological activity scores>0.8 by molecular docking comprises: screening the peptides with predicted biological activity scores>0.8 by semi-flexible molecular docking with cholesterol esterase.

[0018] In some embodiments, the crystal structure information of the cholesterol esterase is obtained from the RCSB database, and the PDB number of the cholesterol esterase is 1F6W; and / or,

[0019] The molecular docking is performed using the software AutoDock 4.2.6; and / or,

[0020] Before the molecular docking, the PDB file of the cholesterol esterase is pre-processed, and the pre-processing includes: deleting water molecules and ligands, and adding hydrogen atoms.

[0021] In some embodiments, the lipid-lowering polypeptide is further screened for one or more of the following:

[0022] In an in vitro activity test, the lipid-lowering polypeptide has an inhibition rate of cholesterol esterase activity of greater than or equal to 30%;

[0023] In an in vitro activity test, the lipid-lowering polypeptide has an inhibition rate of lipase of greater than or equal to 30%;

[0024] In a hyperlipidemic animal model, the lipid-lowering polypeptide showed no toxicity;

[0025] In the hyperlipidemia animal model, the cholesterol-lowering extent of the lipid-lowering polypeptide corresponding drug group was greater than that of the alirocumab positive drug group.

[0026] The present application also provides an application of the above-mentioned lipid-lowering polypeptide for the treatment of lipid-lowering.

[0027] The lipid-lowering polypeptide provided in the present application includes at least one of 116 polypeptides with amino acid sequences such as FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY, etc. The lipid-lowering polypeptide provided in the present application can effectively lower blood lipids and cholesterol without obvious toxic side effects. In the in vitro enzyme activity test, it shows good inhibitory activity on sterol esterase and lipase, and in animal in vivo experiments, it can also effectively lower blood lipids and cholesterol; in toxicity prediction and in vivo animal models, it shows no obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is a schematic diagram of the process of an embodiment of the method for screening blood lipid-lowering polypeptides provided in the present application;

[0030] Figure 2 The docking site and details of the lipid-lowering peptide FFPFF and cholesterol esterase;

[0031] Figure 3 The docking site and details of the lipid-lowering peptide VFFFPF and cholesterol esterase;

[0032] Figure 4 The docking site and details of the lipid-lowering peptide PFFFF and cholesterol esterase;

[0033] Figure 5 The docking site and details of the lipid-lowering peptide AFFFPFF and cholesterol esterase;

[0034] Figure 6 The docking site and details of the lipid-lowering peptide PFFFFY and cholesterol esterase;

[0035] Figure 7 This is a graph showing the inhibition rate of cholesterol esterase activity of the lipid-lowering peptide;

[0036] Figure 8This is a graph showing the inhibition rate of lipid-lowering peptide lipase activity. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application and are not used to limit the present application. In the present application, unless otherwise stated, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present application, the term "including" means "including but not limited to".

[0038] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0039] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0040] In the present application, "at least one" means one or more, and "plurality" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0041] The present application provides a lipid-lowering polypeptide having lipid-lowering activity, wherein the lipid-lowering polypeptide comprises at least one of polypeptides having amino acid sequences of FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY.

[0042] In one embodiment, the lipid-lowering polypeptide comprises PFFFF.

[0043] In one embodiment, the amino acid sequence of the lipid-lowering polypeptide is FFPFF, VFFFPF, PFFFF, AFFFPFF, LFFFPFY. Preferably, the amino acid sequence of the lipid-lowering polypeptide is PFFFF.

[0044] It is understandable that any modification and processing based on the lipid-lowering polypeptide, such as double-end blocking, intermediate residue modification, cyclization modification, etc., are within the protection scope of this patent.

[0045] The lipid-lowering polypeptide provided in the present application can effectively lower blood lipids without obvious toxic side effects. In vitro enzyme activity tests show good inhibitory activity against sterol esterase and lipase, and in vivo animal experiments can also effectively lower blood lipids and cholesterol; toxicity predictions and in vivo animal models show no obvious toxic side effects.

[0046] The present application also provides a method for screening lipid-lowering polypeptides, see Figure 1 , Figure 1 This is a schematic diagram of a process of an embodiment of a method for screening lipid-lowering polypeptides provided in the present application, comprising the following steps:

[0047] Step S1: providing peptides to be screened, predicting the probability of biological activity through the database Peptide Ranker, and screening out peptides with a biological activity prediction score > 0.8;

[0048] Step S2: Using molecular docking to screen the peptides with biological activity prediction scores>0.8 for peptides with lipid-inhibiting activity, and obtaining lipid-lowering polypeptides.

[0049] In this embodiment, lipid-lowering peptides with good lipid-lowering activity can be quickly screened out by performing biological activity screening and molecular docking screening.

[0050] In step S1:

[0051] The peptide to be screened is designed based on the principle of high proportion of hydrophobic amino acids and small molecular weight. Specifically, the peptide to be screened is a polypeptide with 5-7 amino acids, and the proportion of hydrophobic amino acids is ≥80%.

[0052] Among them, the hydrophobic amino acids are also called non-polar amino acids. The side chains of these amino acids are mainly composed of carbon and hydrogen, and it is not easy to form hydrogen bonds with water molecules, so they are hydrophobic. The hydrophobic amino acids include: glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P) and tryptophan (Trp, W).

[0053] In this embodiment, the presence of hydrophobic amino acids in peptides, especially small peptides, will have a positive impact on the lipid-lowering effect of the active peptides, and significantly increase the lipid-lowering effect of the active peptides.

[0054] In step S2:

[0055] The molecular docking screening of the polypeptides with a biological activity prediction score greater than 0.8 may specifically be performed by performing semi-flexible molecular docking screening with cholesterol esterase.

[0056] Furthermore, in a specific embodiment, peptides with a "binding_energy" value of less than -1.22 Kcal for docking with cholesterol esterase molecules were screened to obtain five polypeptides with amino acid sequences of FFPFF, PFFFF, VFFFPF, AFFFPFF, and LFFFPFY.

[0057] The crystal structure information of the cholesterol esterase can be obtained from the RCSB database, and the PDB number of the cholesterol esterase is 1F6W.

[0058] The molecular docking was performed using the software AutoDock 4.2.6. The cholesterol esterase was used as a receptor, and the original PDB file was pre-processed before molecular docking by deleting water molecules, adding hydrogen atoms, and deleting the ligands carried by the original receptor.

[0059] Cholesterol esterase, also known as cholesterol ester hydrolase, is mainly involved in the hydrolysis of cholesterol esters, breaking down cholesterol esters into cholesterol and free fatty acids. Cholesterol esterase plays an important role in cholesterol metabolism, maintaining the balance of cholesterol and fatty acids in the body and playing an important physiological function in regulating cholesterol metabolism.

[0060] In one embodiment, the lipid-lowering polypeptide also predicts the following physicochemical properties:

[0061] (1) ToxinPred website (ToxinPred (iiitd.edu.in)) was used to predict toxicity and steric hindrance;

[0062] (2) Predict hydrophilicity using the ExPasy website (Expasy-ProtParam);

[0063] (3) Predict human intestinal absorption (HIA) through the admetSAR website (admetSAR@LMMD (ecust.edu.cn));

[0064] (4) Predict the isoelectric point through the pepdraw website (PepD http: / / www.pepdraw.com / ).

[0065] The above-mentioned prediction of physicochemical properties is of reference significance for the subsequent development of drug dosage forms. The prediction of physicochemical properties shows that the five lipid-lowering polypeptides have good binding properties with cholesterol esterase, and the total average hydrophilicity is positive, indicating that the five lipid-lowering polypeptides are highly hydrophobic. The two polypeptides FFPFF and PFFFF have good intestinal absorption, while the three polypeptides VFFFPF, AFFFPFF, and LFFFPFY have poor intestinal absorption, which provides support for the subsequent selection of drug dosage forms for these lipid-lowering polypeptides, such as oral or injection dosage forms.

[0066] In one embodiment, the lipid-lowering polypeptide is further screened for one or more of the following:

[0067] (1) In an in vitro activity test, the inhibition rate of the lipid-lowering polypeptide on cholesterol esterase activity is greater than or equal to 30%, such as 32%, 46%, 48%, 51%, 63%, etc.;

[0068] (2) In the in vitro activity test, the lipase inhibition rate of the lipid-lowering polypeptide is greater than or equal to 30%, for example, it can be 37%, 43%, 48%, 51%, 69%, etc.

[0069] (3) In animal models of hyperlipidemia, lipid-lowering peptides showed no toxicity;

[0070] (4) In the hyperlipidemia animal model, the lipid-lowering peptide drug group had a significant lipid-lowering effect, and the cholesterol reduction was greater than that in the alirocumab positive drug group.

[0071] In this embodiment, the cholesterol esterase activity inhibition performance and lipase inhibition performance are tested in vitro and polypeptides with good inhibition performance are screened; through the in vivo hyperlipidemia animal model test, polypeptides with no obvious toxic side effects and capable of significantly reducing blood lipid levels in animals are screened, and the cholesterol-lowering effect is better than that of the commercially available alirocumab.

[0072] As can be seen from the above, the screening method for lipid-lowering peptides provided in this application designs multiple small molecule peptides, predicts the biological activity of the peptides, screens lipid-lowering peptides through molecular docking and in vitro lipid-lowering tests, and performs functional verification, providing technical support for the development of safe and effective lipid-lowering drugs.

[0073] The present application also provides an application of a lipid-lowering polypeptide in a lipid-lowering drug for lipid-lowering treatment.

[0074] The technical scheme and technical effects of the present application are described in detail below through specific embodiments, comparative examples and experimental examples. The following embodiments are only some embodiments of the present application and do not specifically limit the present application. It can be understood that the reagents used in each embodiment, comparative example and experimental example are all commercially available reagents unless otherwise specified.

[0075] Example 1 Short peptide design, activity evaluation and screening of lipid-lowering peptides based on molecular docking technology

[0076] According to the principle of high proportion of hydrophobic amino acids and small molecular weight, peptides with 5-7 amino acids were designed, totaling 116 peptides. These 116 peptides can be synthesized or extracted, and the sequences are identified by LCMS / MS. The amino acid sequences of each peptide are shown in Table 1. Peptide Ranker preliminarily screened the above 116 peptides and screened out peptides with a predicted score of biological activity > 0.8.

[0077] The crystal structure of cholesterol esterase (PDB number: 1F6W) was obtained from the RCSB database (http: / / www.rcsb.org / ), and the peptides with biological activity prediction scores higher than 0.80 were semi-flexibly docked with cholesterol esterase using AutoDock 4.2.6 software. Table 1 shows the active peptide sequences, activity scores and molecular docking scoring results.

[0078] Table 1 Active peptides, activity scores and molecular docking scoring results

[0079]

[0080]

[0081]

[0082]

[0083] The biological activity evaluation scores of the 116 designed peptides were all over 0.8, which is consistent with the characteristics of small peptides with high biological activity. The 116 peptides were semi-flexibly docked with cholesterol esterase, and 5 peptides with relatively stable docking conformations with cholesterol esterase were screened out, with the sequences of FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY.

[0084] The peptide FFPFF was semi-flexibly docked with cholesterol esterase. The key amino acid residues for its interaction with cholesterol esterase are ASP-72 and LYS-243, and the interaction force is hydrogen bond. Figure 2 .

[0085] The peptide VFFFPF was semi-flexibly docked with cholesterol esterase. The other key amino acid residues for cholesterol esterase were ASP-72 and LYS-271, and the interaction force was hydrogen bond. Figure 3 .

[0086] The peptide PFFFF was semi-flexibly docked with cholesterol esterase. The key amino acid residues for its interaction with cholesterol esterase are LYS-271 and LEU-274, and the interaction force is hydrogen bond. Figure 4 .

[0087] The peptide AFFFPFF was semi-flexibly docked with cholesterol esterase. The key amino acid residues that interact with cholesterol esterase are GLY-24, SER-26, ASP-72, GLY-90, and LYS-271. The interaction force is van der Waals force. Figure 5 .

[0088] The peptide LFFFPFY was semi-flexibly docked with cholesterol esterase. The other key amino acid residues for cholesterol esterase were ASP-72, GLN-71, and TYR-270, and the interaction force was hydrogen bond. Figure 6 .

[0089] Example 2 Prediction of physicochemical properties related to lipid-lowering peptides

[0090] According to the potential five lipid-lowering peptides screened in Example 1, the relevant physicochemical properties of the peptides were predicted using relevant software and websites. The toxicity and steric hindrance prediction website is ToxinPred (ToxinPred (iiitd.edu.in)), the total average hydrophilicity prediction website is ExPasy (Expasy-ProtParam), the human intestinal absorption prediction website is admetSAR (admetSAR@LMMD (ecust.edu.cn)), and the isoelectric point prediction website is pepdraw (PepD http: / / www.pepdraw.com / ). As shown in Table 2, the five potential lipid-lowering peptides are non-toxic, the isoelectric point is less than 7, acidic, and the steric hindrance is 0.63-0.64, which is conducive to the binding with cholesterol esterase, and the total average hydrophilicity is positive, indicating that these five peptides are highly hydrophobic. The two polypeptides FFPFF and PFFFF have good intestinal absorbability, while the intestinal absorbability of the three polypeptides VFFFPF, AFFFPFF, and LFFFPFY is poor, and the prediction of physicochemical properties is of reference significance for the subsequent development of pharmaceutical dosage forms.

[0091] Table 2 Prediction of physicochemical properties of five lipid-lowering peptides

[0092]

[0093] Example 3 In vitro lipid-lowering experiment

[0094] The five lipid-lowering peptides screened were prepared by Fmoc solid phase synthesis method, and the purity of the peptides was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry analysis.

[0095] The specific process of cholesterol esterase activity inhibition experiment is as follows:

[0096] The concentrations of the five lipid-lowering peptides were all 2 mg / mL. In a 96-well ELISA plate, 50 μL of sample, 50 μL of 25 μg / mL cholesterol esterase (Sigma) solution, and 50 μL of 10 mM p-nitrophenylbutyrate as substrate were incubated at 25°C for 5 min in a pH 7.0 phosphate buffer (containing 100 mM NaCl, 5.16 mM sodium taurocholate). The absorbance was recorded at 405 nm by an ELISA reader. Simvastatin was used as a positive control and calculated according to formula (1).

[0097]

[0098] In formula (1): A: absorbance of control; B: absorbance of control blank; C: absorbance of sample; D: absorbance of sample blank.

[0099] The specific process of lipase activity inhibition experiment is as follows:

[0100] Add 4 mL of PBS buffer (pH 7.4), 4 mL of polyvinyl alcohol, and olive oil (0.330 g / mL) to the test tube, and add 2 mL (2 mg / mL) of hypolipidemic peptide solution to mix, incubate in a water bath at 37°C for 10 min, then add 1 mL of 2 mg / mL pancreatic lipase (Sigma) solution, react for 15 min, and then add 15 mL of 95% ethanol to terminate the enzyme reaction. Add phenolphthalein dropwise and titrate with standard sodium hydroxide solution until it turns slightly red. In the blank experiment, add pancreatic lipase solution after stopping the reaction. The experiment was processed in parallel 3 times, and calculated according to formula (2).

[0101]

[0102] Results Figure 7 and Figure 8 , Figure 7 This is the inhibition rate of cholesterol esterase activity of lipid-lowering peptide. Figure 8 The figure is the inhibition rate of lipase activity of lipid-lowering peptides. At a concentration of 2 mg / mL, the inhibition rate of cholesterol esterase activity of PFFFF can reach 63%, which is higher than the inhibition activity of the other 4 peptides. At a concentration of 2 mg / mL, the lipase inhibition rate of PFFFF can reach 69%. Comprehensive analysis shows that among the 5 peptides screened by activity evaluation and molecular docking, PFFFF peptide has the best lipid-lowering activity. It can not only inhibit cholesterol esterase, but also effectively inhibit lipase activity, preventing lipase from hydrolyzing fat into glycerol and free fatty acids.

[0103] Example 4 Animal Experiment on Lipid-lowering Peptides

[0104] Establishment of hyperlipidemia animal model: 80 SD rats weighing 80±10 g, 10 blank control rats fed with ordinary feed, and the remaining 70 modeling rats fed with high-fat feed (commercially available, 60% fat content).

[0105] During the modeling period, the blank control group was fed with ordinary feed every day, and the modeling group was fed with high-fat feed. All rats had free access to water and were weighed regularly. After the modeling group was fed with high-fat feed for 4 weeks, the blank control group and the modeling group rats were cut off their tails without fasting and blood was collected. The serum was separated as soon as possible after blood collection to determine the levels of serum TC, TG, LDL-C, and HDL-C. The modeling group was randomly divided into 7 groups (high-fat model group, FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group, and positive drug group). There were no significant differences in TC, TG, LDL-C, and HDL-C among the groups; compared with the blank control group, the differences in TC, TG, and LDL-C in the modeling group were significant, and the modeling was determined to be successful. The administration method was intraperitoneal injection. The solvent system of all peptide groups was 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% normal saline. The alirocumab positive drug group was diluted with normal saline. The injection dose of each group is shown in Table 3:

[0106] Table 3 Dosage table for each group

[0107]

[0108]

[0109] After the single injection, each group continued to be fed for 4 days, then weighed and cut off the tail to collect blood. The blank control group was fed with ordinary feed, and the other groups were fed with high-fat feed. After blood collection, serum was separated as soon as possible to measure serum TC, TG, LDL-C, and HDL-C levels.

[0110] Table 4. Changes in rat body weight

[0111]

[0112] According to the results of body weight in Table 4, after the model was successfully established and the drug was administered for 4 days, there was no significant difference in the body weight of the rats in each group, and the body weight of the blank control group fed with normal feed was slightly lower than that of the other groups fed with high-fat feed. This indicates that the injection of lipid-lowering peptides FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY had no significant effect on the body weight of rats, and the screened lipid-lowering peptides had no obvious toxic side effects as injectable drugs.

[0113] Table 5 Test results of serum indexes of rats injected with drugs

[0114]

[0115]

[0116] As can be seen from the above table, before and after administration, the four indicators of triglyceride, total cholesterol, low-density lipoprotein and high-density lipoprotein blood lipids in the modeling groups (hyperlipidemia model group, FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group, and positive drug group) were significantly different from those in the blank control group, indicating that the hyperlipidemia model was successfully established.

[0117] Elevated low-density lipoprotein is the most important risk factor for cardiovascular disease. As can be seen from the above table, after administration, compared with before administration, the low-density lipoprotein of FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group and positive drug group showed a downward trend. Among them, the low-density lipoprotein of rats in the FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group and positive drug group decreased significantly (P < 0.05), and the decreases were 41.84%, 50.48%, 61.86% and 50.92%, respectively.

[0118] Compared with before administration, the total cholesterol indexes of FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group and positive drug group all decreased, with a decrease of 30.22%, 30.60%, 33.33%, 27.22%, 27.12% and 13.91% respectively. Among them, the cholesterol of FFPFF polypeptide group, VFFFPF polypeptide group and PFFFF polypeptide group decreased significantly (P<0.05). The decrease of cholesterol in each lipid-lowering polypeptide group was higher than that in the positive drug group.

[0119] Although there were no significant differences in triglycerides and high-density lipoprotein before and after administration, the FFPFF polypeptide group, VFFFPF polypeptide group, PFFFF polypeptide group, AFFFPFF polypeptide group, LFFFPFY polypeptide group, and positive drug group also showed corresponding improvements after administration.

[0120] According to the results of animal experiments, the five lipid-lowering peptides screened through activity evaluation and molecular docking can improve blood lipids, among which PFFFF peptide has the best lipid-lowering effect, and its effect in reducing low-density lipoprotein and total cholesterol is better than the positive drug alirocumab.

[0121] The lipid-lowering peptides screened in the present application can effectively reduce the levels of total cholesterol and low-density lipoprotein in hyperlipidemic rats, improve the levels of triglycerides and high-density lipoprotein, and the lipid-lowering effect of the PFFFF polypeptide is better than that of the positive drug alirocumab on the market.

[0122] The lipid-lowering polypeptides and their screening methods and applications provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the methods and core ideas of the present application. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A lipid-lowering polypeptide, characterized in that: The lipid-lowering polypeptide(s) includes one or more of the following 116 polypeptides with the following amino acid sequences: FPFFF, FFPFF, FFFFP, PFFFF, FFFPFF, FFFPFW, FFFPFL, FFFPFG, FFFPFY, FFFPFI, FFFPFA, FFFPFV, FFFF PF, WFFFPF, LFFFPF, GFFFPF, YFFFPF, IFFFPF, AFFFPF, VFFFPF, FFFF PFF, FFFF PF W, FFFF PFL, FFFF PFG, FFFF PFY, FFFF PFI, FFFF PFA, FFFF PFV, FFFFFPF, FWFFFPF, FLFFFPF, FGFFFPF, FYFFFPF, FIFFFPF, FAFFFPF, FVFFFPF, FFFPFFF, FFFPFWF, FFFPFLF, FFFPFGF, FFFPFYF, FFFP FIF, FFFP AFF, FFFP FVF, FFFF PFF, WFFFPFF, LFFFPFF, GFFFPFF, YFFFPFF, IFFFPFF, AFFFPFF, VFFFPFF, FFFPFFW, FFFPFWW, FFFPFLW, FFFPFGW, FFFPFYW, FFFPFIW, FFFPFAW, FFFPFVW, FFFF PF W, WFFFPFW, LFFFPFW, GFFFPFW, YFFFPFW, IFFFPFW, AFFFPFW, VFFFPFW, WFFFPFF, WFFFPFW, WFFFPFL, WFFFPFG, WFFFPFY, WFFFPFI, WFFFPFA, WFFFPFV, WFFFFPF, WWFFFPF, WLFFFPF, WGFFFPF, WYFFFPF, WIFFFPF, WAFFFPF, WVFFFPF, WFFFPFL, FFFPFFL, FFFPFWL, FFFF PFL, LWFFFPF, LFFFPFF, LFFFPFW, LFFFFPF, GFFFPFL, AFFFPFL, FFFP FLL, YFFFPFL, LGFFFPF, LFFFPFL, LFFFPFL, LYFFFPF, FFFPFGL, FFFPFYL, IFFFPFL, LAFFFPF, FFFP FIL, VFFFPFL, LIFFFPF, LFFFPFG, LLFFFPF, LFFFPFY, FFFP FAL, FFFP FVL, LFFFPFI, LVFFFPF, LFFFPFA, LFFFPFV.

2. The lipid-lowering polypeptide according to claim 1, characterized in that The lipid-lowering polypeptide(s) includes one or more of the polypeptides with the amino acid sequences of FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY.

3. The lipid-lowering polypeptide according to claim 2, characterized in that The amino acid sequence of the lipid-lowering polypeptide is FFPFF, VFFFPF, PFFFF, AFFFPFF, and LFFFPFY.

4. The lipid-lowering polypeptide according to claim 2, characterized in that The lipid-lowering polypeptide includes PFFFF; or, the amino acid sequence of the lipid-lowering polypeptide is PFFFF.

5. The method for screening lipid-lowering polypeptides according to any one of claims 1 to 4, characterized in that: include: Provide the peptides to be screened, predict the probability of biological activity through the Peptide Ranker database, and screen out peptides with a biological activity prediction score > 0.8; The peptides with biological activity prediction scores>0.8 were screened out by molecular docking to obtain lipid-lowering peptides.

6. The screening method according to claim 5, characterized in that The peptide segment to be screened is a polypeptide having 5-7 amino acids; and / or, The proportion of hydrophobic amino acids in the peptide to be screened is ≥80%; and / or, The providing of peptide segments to be screened includes: providing 116 peptide segments to be screened.

7. The screening method according to claim 5, characterized in that The method of screening the peptides with predicted biological activity scores>0.8 by using molecular docking includes: screening the peptides with predicted biological activity scores>0.8 by semi-flexible molecular docking with cholesterol esterase.

8. The screening method according to claim 7, characterized in that The crystal structure information of the cholesterol esterase is obtained from the RCSB database, and the PDB number of the cholesterol esterase is 1F6W; and / or, The molecular docking is performed using the software AutoDock 4.2.6; and / or, Before the molecular docking, the PDB file of the cholesterol esterase is pre-processed, and the pre-processing includes: deleting water molecules and ligands, and adding hydrogen atoms.

9. The screening method according to any one of claims 4 to 8, characterized in that: The lipid-lowering polypeptide is further screened for one or more of the following: In an in vitro activity test, the lipid-lowering polypeptide has an inhibition rate of cholesterol esterase activity of greater than or equal to 30%; In an in vitro activity test, the lipid-lowering polypeptide has an inhibition rate of lipase of greater than or equal to 30%; In a hyperlipidemic animal model, the lipid-lowering polypeptide showed no toxicity; In the hyperlipidemia animal model, the cholesterol-lowering extent of the lipid-lowering polypeptide corresponding drug group was greater than that of the alirocumab positive drug group.

10. Use of the lipid-lowering polypeptide according to any one of claims 1 to 4 or the lipid-lowering polypeptide screened by the screening method according to any one of claims 5 to 9, characterized in that: Used for lipid-lowering treatment.

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