Polypeptide for regulating low-density lipoprotein receptor-associated protein (LRP) and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a method for regulating the activity of low-density lipoprotein receptor-associated protein (LRP) and a medicine application of the low-density lipoprotein receptor-associated protein (LRP). The polypeptide has a unique amino acid sequence, and comprises the amino acid sequences of SEQ. NO: 1: Ala-Pro-Glu-Met-Thr (APEMT), SEQ. NO: 2: Cys-His-Val-Asp-Asn (CHVDN), SEQ. NO: 3: Gly-Leu-Ser-Arg-Phe (GLSRF) and SEQ. NO: 4: Tyr-Trp-Lys-His-Glu (YWKHE), and the novel polypeptide can effectively regulate the activity of LRP, improve lipid metabolism and reduce the blood fat level. Meanwhile, the polypeptide has no obvious toxicity under high dosage, has no obvious adverse effect on important organs such as liver, kidney and the like after being used for a long time, and has relatively high safety and reliability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and mainly relates to the research and development of a novel polypeptide and its application in the treatment of cardiovascular diseases. Background Art

[0002] The severe current situation of hyperlipidemia: Hyperlipidemia is a common metabolic disease, mainly manifested as abnormal elevation of lipid components such as cholesterol and triglyceride in the blood. Long-term hyperlipidemia can lead to the occurrence of cardiovascular diseases such as atherosclerosis, coronary heart disease, and stroke, seriously threatening human health. At present, the treatment methods of hyperlipidemia mainly include diet control, exercise, drug treatment, etc. However, the effects of diet and exercise therapies are limited and require long-term adherence; although drug treatment can effectively reduce blood lipid levels, there are also some side effects, such as abnormal liver function, muscle pain, etc. Therefore, it is of great practical significance to find a safer and more effective treatment method.

[0003] The development of molecular biology and proteomics brings new opportunities for the treatment of hyperlipidemia: In recent years, with the rapid development of molecular biology and proteomics, researchers have begun to focus on developing novel hyperlipidemia treatment methods by targeting specific proteins. This method has high specificity and effectiveness, can precisely treat the pathogenesis of hyperlipidemia, and reduce the side effects of traditional treatment methods.

[0004] Biological characteristics of key proteins: Low-density lipoprotein receptor-related protein (LRP) is an important lipid metabolism regulatory protein, widely present in various tissue cells. It plays an important role in lipid metabolism, mainly including participating in cholesterol transport, regulating triglyceride metabolism, etc. LRP consists of multiple domains and has a complex structure and function. Under normal physiological conditions, LRP interacts with multiple lipid metabolism-related proteins to maintain the balance of lipid metabolism. When lipid metabolism is disordered, the expression and function of LRP may change, leading to the occurrence of hyperlipidemia.

[0005] Important regulatory role of LRP in hyperlipidemia: Cholesterol transport: LRP can bind to low-density lipoprotein (LDL), promote the endocytosis and degradation of LDL, thereby reducing the cholesterol level in the blood. In addition, LRP can also participate in the metabolism of high-density lipoprotein (HDL), regulate the reverse transport of cholesterol, and transport cholesterol in peripheral tissues to the liver for metabolism.

[0006] Triglyceride metabolism: LRP can interact with triglyceride metabolism-related proteins to regulate the synthesis and decomposition of triglycerides. For example, LRP can promote the decomposition of triglycerides in adipocytes and reduce the triglyceride level in the blood.

[0007] The potential of LRP as a therapeutic target for hyperlipidemia: Development of LRP modulators: Since LRP plays an important role in the occurrence and development of hyperlipidemia, the development of LRP modulators has become a potential therapeutic strategy for hyperlipidemia. Currently, some studies have attempted to develop LRP modulators, such as small molecule compounds, antibodies, etc. These modulators can improve lipid metabolism and reduce blood lipid levels by regulating the expression and function of LRP.

[0008] Possibility of combination therapy: In addition to using LRP modulators alone, the combination of LRP modulators with other hyperlipidemia drugs can also be considered to improve the therapeutic effect. For example, LRP modulators can be combined with statins, fibrates, etc. to exert a synergistic therapeutic effect. Challenges and future prospects: Although LRP has great potential in the treatment of hyperlipidemia, there are still some challenges at present.

[0009] Specificity and safety issues: Most of the currently developed LRP modulators lack specificity and may have adverse effects on other normal cells and tissues. In addition, the safety of LRP modulators needs to be further evaluated, especially the side effects that may occur during long-term use.

[0010] Complexity of the mechanism of action: The mechanism of action of LRP in hyperlipidemia is very complex, involving the regulation of multiple signal transduction pathways and lipid metabolism-related proteins. At present, the specific mechanism of action of LRP in hyperlipidemia is not fully understood and further in-depth research is needed.

[0011] Feasibility of clinical application: Although LRP modulators have shown certain therapeutic potential in animal experiments and cell experiments, their feasibility and effectiveness need to be further verified in clinical applications. In addition, the administration method, dosage, and treatment course of LRP modulators also need to be optimized to improve the therapeutic effect and reduce side effects.

[0012] It is necessary to research and develop more specific and safe LRP modulators, deeply study the mechanism of action of LRP, and conduct large-scale clinical trials to provide new ideas and methods for the treatment of hyperlipidemia. Summary of the Invention

[0013] (I) Structure of the novel polypeptide The novel polypeptide provided by the present invention is composed of the following four amino acid sequences: SEQ.NO 1: Ala - Pro - Glu - Met - Thr (APEMT); SEQ.NO 2: Cys - His - Val - Asp - Asn (CHVDN); SEQ.NO 3: Gly - Leu - Ser - Arg - Phe (GLSRF); SEQ.NO 4: Tyr - Trp - Lys - His - Glu (YWKHE).

[0014] These sequences were designed by conducting in - depth research on the structure and function of the target protein LRP and combining binding affinity analysis.

[0015] (II) Mechanism of action of the novel polypeptides Regulating LRP activity The novel polypeptides can specifically bind to LRP, thereby regulating its activity. Specifically, the amino acids in the APEMT sequence have specific chemical properties, such as the hydrophobicity of Ala, the rigidity of Pro, the negative charge of Glu, the thioether group of Met, and the hydroxyl group of Thr. These properties contribute to forming complementary interactions with specific binding regions of LRP, thus regulating the activity of LRP. Similarly, the amino acids in the CHVDN, GLSRF, and YWKHE sequences can also interact with LRP and affect its function.

[0016] Improving lipid metabolism By regulating the activity of LRP, the novel polypeptides can improve lipid metabolism. LRP is involved in the transport and metabolism of cholesterol and triglycerides and plays an important role in the balance of lipid metabolism. The novel polypeptides can regulate the expression and function of LRP, promote the reverse transport of cholesterol and the decomposition of triglycerides, thereby reducing the lipid levels in the blood.

[0017] Furthermore, the novel polypeptides also include TQKIAHPGSFLYDVQNC, VQMRDSHKPCM WTAKYIG, GDTSHRELPMYNKIVAC for regulating the activity of low - density lipoprotein receptor - related protein (LRP).

[0018] Advantages of the invention: High efficiency The novel polypeptides can significantly improve the functional state of the target protein LRP and effectively regulate physiological indexes related to lipid metabolism, such as reducing cholesterol and triglyceride levels, etc.

[0019] Safety Verified by in - vitro and in - vivo experiments, the novel polypeptides have no obvious toxicity at high doses and have no obvious adverse effects on important organs such as the liver and kidneys after long - term use.

[0020] Specificity The novel polypeptide has a high specific binding ability to the target protein LRP, reducing interference with other irrelevant proteins and lowering the risk of potential side effects. Detailed implementation methods

[0021] Example 1

[0022] Example 1 (I) Synthesis of the polypeptide Selection of amino acid monomers Amino acid monomers with Boc protecting groups are used to ensure the activity and stability of amino acids during synthesis.

[0023] Solid-phase synthesis Amino acid monomers are sequentially linked to the solid-phase carrier. After each coupling reaction, the Boc group is removed through a deprotection step. A step-by-step synthesis method is adopted to ensure the accuracy and purity of the polypeptide.

[0024] Cleavage and purification After all amino acids are linked, they are cleaved from the solid-phase carrier to obtain the crude polypeptide. Its purity and molecular weight are confirmed by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Multiple purification methods, such as reverse-phase high-performance liquid chromatography and ion-exchange chromatography, are used to improve the purity of the polypeptide.

[0025] (II) Structure verification experiments Mass spectrometry analysis Instrument: A high-resolution mass spectrometer (such as MALDI-TOF MS) is used for analysis.

[0026] Sample preparation: The synthesized peptide segments are respectively dissolved in a mixed solvent of methanol and water to prepare a solution with an appropriate concentration.

[0027] Experimental results: The mass spectrometry analysis results show that the molecular weights of the peptide segments are consistent with the theoretical values, further confirming the correctness of their structures. By comparing the difference between the measured molecular weight and the theoretical molecular weight, the synthesis accuracy of the polypeptide is evaluated.

[0028] Circular dichroism (CD) analysis Instrument: A circular dichroism spectrometer is used for analysis.

[0029] Sample preparation: The synthesized peptide segments are respectively dissolved in phosphate buffer to prepare a solution with an appropriate concentration.

[0030] Experimental results: The circular dichroism analysis results show that the peptide segments have specific secondary structure characteristics, which are consistent with the expected structures. The secondary structure of the polypeptide, such as α-helix and β-sheet, is analyzed to understand its structural stability.

[0031] (III) Identification and related research of target protein LRP Identification of LRP LRP was identified in hyperlipidemia-related tissues by proteomics and bioinformatics methods. Immunohistochemistry, Western blot and other techniques were used to verify the expression of LRP in tissues of hyperlipidemia patients.

[0032] Determination of binding site The binding sites of the novel polypeptide and LRP were determined by molecular docking technology. Computer simulation software was used to predict the binding mode and affinity of the polypeptide and LRP, providing a basis for further optimizing the polypeptide structure.

[0033] (IV) In vitro activity test Cell culture (1) Cell line: human hepatocyte cell line (HepG2 cells).

[0034] (2) Culture conditions: DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 37°C, 5% CO 2 .

[0035] Peptide treatment (1) Each synthesized polypeptide was dissolved in DMSO to prepare solutions with different concentrations.

[0036] (2) Polypeptides with different concentrations were added to the HepG2 cell culture medium and incubated for 24 hours.

[0037] Experimental results (1) LRP activity: Its activity status was reflected by detecting cholesterol uptake and triglyceride metabolism-related indicators mediated by LRP. The results showed that each polypeptide significantly inhibited the activity of LRP. The specific data are as follows: (2) Lipid metabolism indicators: Cholesterol level: The cholesterol content in cells was detected by a kit. The results showed that each polypeptide significantly reduced the cholesterol level in HepG2 cells. The specific data are as follows: Triglyceride level: The triglyceride content in cells was detected by a kit. The results showed that each polypeptide significantly reduced the triglyceride level in HepG2 cells. The specific data are as follows: (V) Verification in animal models Animal model (1) Model: hyperlipidemia mouse model (prepared by feeding a high-fat diet).

[0038] (2) Experimental group: each polypeptide treatment group.

[0039] (3) Control group: Normal saline treatment group.

[0040] (4) Positive control group: Treatment group with common lipid-lowering drugs (such as atorvastatin).

[0041] Administration method Each polypeptide or normal saline was administered daily by gavage for six consecutive weeks.

[0042] Experimental results (1) Blood lipid levels: The blood lipid levels of mice, including cholesterol, triglyceride, etc., were regularly detected. The results showed that the blood lipid levels in each polypeptide treatment group were significantly lower than those in the control group and the atorvastatin treatment group. The specific data are as follows: (2) Liver function indicators: By detecting the liver function indicators of mice, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), etc., the effect of polypeptides on the liver was evaluated. The results showed that there was no significant difference in the liver function indicators between each polypeptide treatment group and the control group, indicating that the polypeptides had no obvious adverse effects on the liver. The specific data are as follows: (6) Safety assessment Acute toxicity test By administering a single large dose of drug, the survival rate and behavioral performance of mice were observed. The results showed that each polypeptide had no obvious toxicity at high doses. The specific data are as follows: Long-term toxicity test By administering the drug continuously for eight weeks, indicators such as the body weight, blood routine, liver and kidney functions of mice were observed. The results showed that each polypeptide had no obvious adverse reactions under long-term use. The specific data are as follows: Data table of comparative synergistic test Example 2 Small molecule peptide derived from Pholidota chinensis Lindl. extract Small molecule peptide sequence: SEQ.NO 5: Thr - Gln - Lys - Ile - Ala - His - Pro - Gly - Ser - Phe - Leu - Tyr - Asp - Val - Gln - Asn - Cys (TQKIAHPGSFLYDVQNC) Source: Pholidota chinensis Lindl. is a plant of the genus Pholidota in the Orchidaceae family, which is mostly distributed in the mountainous areas of southern China and is used for moistening the lungs and relieving cough in traditional medicine.

[0043] Extraction method: Collect fresh whole plants of Pholidota chinensis, wash them, dry them at 60 °C until constant weight, and pulverize them through an 80-mesh sieve. Using ultrasonic-assisted complex enzymatic hydrolysis method, add the powder to phosphate buffer solution with pH 7.6 according to the solid-liquid ratio of 1:25 (g / mL), then add neutral protease with a mass fraction of 1% and cellulase with a mass fraction of 0.6%, and carry out enzymatic hydrolysis for 3.5 h under the ultrasonic power of 40 kHz, with the temperature controlled at 37 °C. After the enzymatic hydrolysis is completed, heat at 90 °C for 12 min to inactivate the enzyme. Subsequently, centrifuge at 6000 r / min for 20 min and collect the supernatant. The supernatant is ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da to remove macromolecular impurities. The ultrafiltered filtrate is preliminarily separated by Sephadex G-20 dextran gel, using 0.1 M sodium chloride solution as the eluent, with a flow rate of 0.4 mL / min. Collect the elution peak containing small molecule peptides, and then identify the sequence of this small molecule peptide by high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) technology.

[0044] Synergistic effect: Combine TQKIAHPGSFLYDVQNC with the novel polypeptides (APEMT, CHVDN, GLSRF, YWKHE) of the present invention. In in vitro cell experiments, when using TQKIAHPGSFLYDVQNC alone, the reduction rate of cholesterol level in human hepatocyte line (HepG2 cells) is 15%, and when using the novel polypeptide of the present invention alone (taking APEMT as an example), the reduction rate is 20%. When the two are combined, the reduction rate of cholesterol level in HepG2 cells reaches 40%, showing a significant synergistic effect. In animal experiments, for the hyperlipidemia mouse model, using TQKIAHPGSFLYDVQNC alone can reduce cholesterol by 0.5 mmol / L, using APEMT alone can reduce 0.7 mmol / L, and after combination, cholesterol is reduced by 1.5 mmol / L, far exceeding the sum of the effects of the two used alone, further verifying its synergistic characteristics in improving lipid metabolism.

[0045] Example 3 Small molecule peptide derived from the extract of Zanthoxylum nitidum Small molecule peptide sequence: SEQ.NO 6: Val - Gln - Met - Arg - Asp - Ser - His - Lys - Pro - Cys - Met - Trp - Thr - Ala - Tyr - Lys - Ile - Gly (VQMRDSHKPCMWTAKYIG) Source: Zanthoxylum nitidum is a plant of the genus Zanthoxylum in the Rutaceae family, and its roots and stems are commonly used in medicine.

[0046] Extraction method: Take the dried roots of Zanthoxylum nitidum, crush them and sieve through a 100-mesh sieve. Use supercritical CO 2 extraction combined with aqueous two-phase extraction technology. The supercritical CO 2 extraction conditions are: pressure 32 MPa, temperature 48 °C, CO 2 flow rate 22 L / h, extraction time 2.2 h. Collect the extract, dissolve it with an appropriate amount of water, mix it with a 25% (mass fraction) PEG 6000 and an 18% ammonium sulfate solution in a volume ratio of 1:1:1 to form an aqueous two-phase system. After shaking and mixing evenly, centrifuge at 3500 r / min for 12 min, and collect the upper phase rich in small molecule peptides. Perform ion exchange chromatography separation on the upper phase, select a strong cation exchange resin, and perform gradient elution with sodium chloride solutions of different concentrations at a flow rate of 1.2 mL / min. Collect the eluate containing the target peptide, and further purify it by preparative liquid chromatography to determine the sequence of this small molecule peptide.

[0047] Synergistic effect: In in vitro experiments, the reduction rate of intracellular triglyceride levels in HepG2 cells by using VQMRDSHKPCM WTAKYIG alone was 18%, and the reduction rate by using CHVDN of the present invention alone was 22%. When the two were combined for treatment, the reduction rate of intracellular triglyceride levels in HepG2 cells reached 45%, showing an obvious synergistic effect. In the experiment of hyperlipidemia mouse model, using VQMRDSHKPCM WTAKYIG alone could reduce triglyceride by 0.4 mmol / L, using CHVDN alone could reduce it by 0.5 mmol / L, and after combined use, triglyceride was reduced by 1.2 mmol / L, indicating that the combined use has a significant synergistic effect in regulating LRP activity and improving lipid metabolism, providing a better treatment plan for hyperlipidemia.

[0048] Example 4 Small molecule peptide derived from Phyla nodiflora extract Small molecule peptide sequence: SEQ.NO 7: Gly - Asp - Thr - Phe - Ser - His - Arg - Glu - Leu - Pro - Met - Tyr - Asn - Lys - Ile - Val - Ala - Cys (GDTSHR ELPMYNKIVAC) Source: Phyla nodiflora is a plant of the genus Phyla in the Verbenaceae family. It grows mostly by streams and roadsides and has certain medicinal value. There have been no reports on its related research in regulating LRP activity.

[0049] Extraction method: Collect the whole plant of Lippia nodiflora, wash it, freeze-dry it, and pulverize it through a 120-mesh sieve. Using microwave-assisted extraction combined with affinity chromatography technology, add the powder to 60% ethanol solution at a solid-liquid ratio of 1:22 (g / mL), extract for 25 min at a microwave power of 600 W, and control the temperature at 55 °C. After extraction, centrifuge at 4500 r / min for 18 min, collect the supernatant, and concentrate it under reduced pressure until the alcohol smell disappears. Extract the concentrated solution with an equal volume of petroleum ether 5 times, discard the upper petroleum ether layer to remove lipophilic impurities. Then, extract the lower aqueous phase with an equal volume of ethyl acetate 5 times, collect the ethyl acetate layer, and concentrate it under reduced pressure. The concentrate is preliminarily separated by affinity chromatography. Select an affinity packing material that can specifically bind to small peptides, use 0.1 M phosphate buffer (pH 7.5) as the eluent, and the flow rate is 0.9 mL / min. Collect the eluent containing the target peptide, and then identify it by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) technology to determine the sequence of the small peptide.

[0050] Synergistic effect: In vitro, when using GDTSHR ELPMYNKIVAC alone, the inhibition rate of LRP activity (measured by the amount of cholesterol uptake mediated) is 25%, and when using GLSRF of the present invention alone, the inhibition rate is 30%. When used in combination, the inhibition rate of cholesterol uptake mediated by LRP reaches 60%, showing a synergistic effect. In animal experiments, for the hyperlipidemia mouse model, when using GDTSHR ELPMYNKIVAC alone, the comprehensive blood lipid index (the sum of cholesterol and triglyceride) can be reduced by 1.0 mmol / L, when using GLSRF alone, it can be reduced by 1.2 mmol / L, and after combined use, the comprehensive blood lipid index is reduced by 2.5 mmol / L, proving that the combined use has a synergistic effect in improving lipid metabolism and providing a new idea for the treatment of hyperlipidemia. In order to verify the effect of the novel polypeptide of the present invention in the treatment of hyperlipidemia, a series of experiments were carried out. The experimental results show that the novel polypeptide can effectively regulate the activity of LRP, improve lipid metabolism, and reduce blood lipid levels. At the same time, the polypeptide has no obvious toxicity at high doses and has no obvious adverse effects on important organs such as the liver and kidneys after long-term use, and has high safety and reliability.

Claims

1. A polypeptide for regulating low-density lipoprotein receptor-related protein (LRP), characterized in that: It comprises the amino acid sequence SEQ.NO 1: Ala - Pro - Glu - Met - Thr (APEMT), SEQ.NO 2: Cys - His - Val - Asp -Asn (CHVDN), SEQ.NO 3: Gly - Leu - Ser - Arg - Phe (GLSRF), SEQ.NO 4: Tyr - Trp- Lys - His - Glu (YWKHE).

2. The novel polypeptide according to claim 1, characterized in that The novel polypeptides also include TQKIAHPGSFLYDVQNC, VQMRDSHKPCM WTAKYIG, and GDTSHR ELPMYNKIVAC for regulating the activity of low-density lipoprotein receptor-related protein (LRP).

3. The novel polypeptide according to claim 1, characterized in that The novel polypeptide improves lipid metabolism by binding to LRP and is used for treating hyperlipidemia.

4. The novel polypeptide according to claim 1, characterized in that The novel polypeptide is prepared by solid phase synthesis, which includes sequentially connecting amino acid monomers with Boc protective groups to a solid phase carrier, followed by cutting and purification.

5. The novel polypeptide according to claim 1, characterized in that The structure of the novel polypeptide was verified by mass spectrometry analysis and circular dichroism analysis, and its molecular weight was consistent with the theoretical value, and it had specific secondary structure characteristics.

6. The novel polypeptide according to claim 1, characterized in that In in vitro cell experiments, the novel polypeptide can significantly inhibit the activity of LRP and reduce the levels of cholesterol and triglycerides in human liver cell lines (HepG2 cells).

7. The novel polypeptide according to claim 1, characterized in that Application of novel peptides in the preparation of drugs for treating hyperlipidemia.