A novel polypeptide drug and its preparation method and application

Abstract

The present invention discloses a novel polypeptide drug, a preparation method and an application, belonging to the field of biomedicine technology. The polypeptide drug comprises a targeting module R4, a long retention module R3, a self-assembly module R2 and a hydrophobic module R1 connected in sequence by amide bonds; wherein R4 is derived from a PCSK9 targeting peptide; R3 is derived from polyethylene glycol distearate; R2 is derived from a multi-hydrogen bond polypeptide sequence; and R1 is derived from a C10-C20 alkyl carboxylic acid. The present invention utilizes the characteristic that the novel polypeptide drug specifically recognizes the PCSK9 protein, and under the action of multiple hydrogen bonds, a nanofiber network is formed on the surface of the PCSK9 protein, and the PCSK9 protein is better bound by the synergistic action of multivalent bonds, and the binding of the PCSK9 protein to LDLR is competitively inhibited, and the degradation of LDLR is reduced, thereby playing a role in preventing and treating hypercholesterolemia, which provides a new idea and method for improving or treating hypercholesterolemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel polypeptide drug and a preparation method and application thereof. Background Art

[0002] Numerous genetic and clinical studies have demonstrated that high levels of circulating low-density lipoprotein cholesterol (LDL-C) are associated with the development and progression of atherosclerotic cardiovascular disease (ASCVD), the leading cause of all deaths worldwide. Therefore, scientists in academia and industry have placed great emphasis on developing therapeutic approaches to lower LDL cholesterol levels to treat hypercholesterolemia. For many years, statins have been the first-line treatment for lowering LDL levels and cardiovascular events. Although they are highly effective, they have certain limitations due to side effects such as muscle toxicity, gastrointestinal symptoms, and elevated liver enzymes, and statins often fail to reduce cholesterol levels to the expected level (~50%) in a large proportion of patients. In the first decade of this century, proprotein convertase subtilisin / kexin type 9 (PCSK9) has become a new target for lowering low-density lipoprotein levels. For those who are intolerant to statins, those who are contraindicated with statins, or those whose LDL-C levels are still poorly controlled after receiving the maximum tolerated dose, PCSK9 inhibitors make it possible to achieve blood lipid standards, which is undoubtedly good news for these people. Due to its good lipid-lowering effect and low frequency of medication, it has become a new option for the treatment of hypercholesterolemia.

[0003] A compound that can reduce the amount of PCSK9 protein and a preparation method thereof, with publication number CN104529910B, provides an optically active dibenzylamine derivative that can reduce the amount of PCSK9 protein, increase the amount of LDL receptors, and also show an excellent effect of lowering LDL cholesterol in the blood, so it can be used as an effective ingredient for lipid-lowering drugs.

[0004] Publication No. CN105431447A discloses a small molecule regulator of PCSK9 and a method of using the same. The small molecule in the application can inhibit PCSK9, and its effect in combination with statins that increase PCSK9 expression was evaluated. The results showed that statins + PCSK9 inhibitors have a better therapeutic effect on high LDL-C.

[0005] However, overall, there are still few peptide drugs that inhibit PCSK9 to treat hypercholesterolemia. Therefore, it is very meaningful to develop a PCSK9 targeted drug with greater advantages in terms of cost and safety. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a novel polypeptide drug and a preparation method and application thereof, so as to solve the problem that there are few polypeptide drugs for treating hypercholesterolemia and hypercholesterolemia.

[0007] To achieve the above object, the present invention provides the following technical solutions: a novel polypeptide drug, the polypeptide drug comprising a targeting module R4, a long retention module R3, a self-assembly module R2 and a hydrophobic module R1 connected in sequence by amide bonds;

[0008] Among them, R4 comes from the PCSK9 targeting peptide; R3 comes from polyethylene glycol distearate; R2 comes from a multiple hydrogen bond polypeptide sequence; and R1 comes from a C10-C20 alkyl carboxylic acid.

[0009] Furthermore, the alkyl chain of the alkyl carboxylic acid is a straight chain, the carbon number of the alkyl chain of the alkyl carboxylic acid is 16, and the R1 is n-hexadecanoic acid.

[0010] Furthermore, the multiple hydrogen-bonding polypeptide sequence includes FFVLK or DFFPL.

[0011] Furthermore, the polyethylene glycol distearate is PEG-4 distearate.

[0012] Furthermore, the PCSK9 targeting peptide is a small peptide segment Pep2-8: TVFTSWEEYLDWV.

[0013] Furthermore, a method for preparing a novel polypeptide drug is provided, wherein the polypeptide drug for treating hypercholesterolemia is synthesized by solid phase synthesis using amino acids with protected terminal amino groups and side chain amino groups, and C10-C20 alkyl carboxylic acids as raw materials.

[0014] Furthermore, the active ingredients of the drug are n-hexadecanoic acid, a polypeptide sequence FFVLK, PEG4 and a polypeptide sequence TVFTSWEEYLDWV connected in sequence through amide bonds.

[0015] Furthermore, the dosage form of the drug is any pharmaceutically acceptable dosage form; the drug also includes pharmaceutically acceptable pharmaceutical excipients, and the pharmaceutical excipients include any one of fillers, excipients, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0016] Furthermore, a novel peptide drug is used to treat hypercholesterolemia.

[0017] Furthermore, a novel peptide drug is used to treat hypercholesterolemia.

[0018] The beneficial effects of the present invention are:

[0019] The present invention utilizes the characteristic of the novel polypeptide drug that specifically recognizes the PCSK9 protein. Under the action of multiple hydrogen bonds, a nanofiber network is formed on the surface of the PCSK9 protein. The PCSK9 protein is better bound by the synergistic action of multivalent bonds, thereby competitively inhibiting the binding of the PCSK9 protein to LDLR and reducing the degradation of LDLR, thereby playing a role in preventing and treating hypercholesterolemia. This provides a new idea and new method for improving or treating hypercholesterolemia.

[0020] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art may be taught from the practice of the present invention. The objectives and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the purpose, technical solution and beneficial effects of the invention clearer, the present invention provides the following drawings for illustration:

[0022] Figure 1 is the chemical structural formula of the polypeptide drug for treating hypercholesterolemia prepared in Example 1;

[0023] Figure 2 This is a mass spectrometry characterization diagram of the polypeptide drug for treating hypercholesterolemia prepared in Example 1;

[0024] Figure 3 This is a scanning electron microscope characterization image after NSP and PCSK9 protein were co-incubated in Example 2;

[0025] Figure 4 This is a characterization diagram of the fluorescence emission spectrum after co-incubation of NSP and PCSK9 protein in Example 2;

[0026] Figure 5 This is a laser confocal microscope result of the binding of NSP labeled with Cy5 and FITC-labeled PCSK9 protein in Example 3;

[0027] Figure 6 The Western blot results of LDLR after NSP and Pep2-8 were co-incubated with HepG2 cells in Example 4;

[0028] Figure 7 The figure shows the protein blotting results of liver LDLR in C57BL / 6 mice treated with PBS and NSP respectively in Example 5. DETAILED DESCRIPTION

[0029] Example 1

[0030] Example 1 is used to construct a novel polypeptide drug (hereinafter referred to as NSP) for treating hypercholesterolemia. The chemical formula of the self-assembled material for preventing and treating hypercholesterolemia is:

[0031] CH3(CH2) 14 CO-FFVLK-PEG4-TVFTSWEEYLDWV

[0032] The preparation method of the polypeptide drug is: using the polypeptide solid phase synthesis method, selecting Fmoc-protected valine with a modification density of 0.33 of Wang resin as the starting material, and synthesizing the NSP polypeptide by adding amino acids one by one in sequence.

[0033] The obtained peptide was characterized by mass spectrometry. Figure 2 As shown by Figure 2 It can be seen that the main peak of the mass spectrum is consistent with the molecular weight of the designed polypeptide material, which indicates that the polypeptide with the above structure was successfully synthesized.

[0034] Example 2

[0035] Example 2 is used to prepare a polypeptide solution:

[0036] The NSP polypeptide was dissolved in DMSO solvent (concentration of 20 mM), 10 μL of the above solution was added to 990 μL of deionized water, pipetted and mixed, and vortexed for 30 seconds to obtain a polypeptide solution.

[0037] After the peptide solution was diluted with deionized water to a concentration of 30 μM, PCSK9 protein (protein concentration was 1×10-7 M) was added, and the morphological transformation of the peptide was characterized by scanning electron microscopy at 0 h, 4 h, 8 h, 12 h, and 24 h. The results are as follows: Figure 3 As shown by Figure 3 It can be seen that the polypeptide changes from granular to fibrous over time.

[0038] Fluorescence characterization was performed after incubation with Thioflavin T fluorescent probe for 2 h at 0 h, 0.5 h, 2 h, 4 h, and 6 h. The characterization results are shown in Figure 4 As shown, the fluorescence signal at about 482 nm increases with time, indicating that the antiparallel β-folded fibrils formed by the THT-binding polypeptide increase, proving the existence of nanofibers.

[0039] Example 3

[0040] Example 3 is used to explore the binding of peptide drugs to PCSK9:

[0041] The NSP prepared in Example 1 was labeled with Cy5 and then co-incubated with FITC-labeled PCSK9 protein.

[0042] After 12 h of co-incubation, laser confocal imaging was performed to observe the co-localization of the two materials.

[0043] Test results such as Figure 5 As shown, the Pearson correlation coefficient between the two is 0.7974, indicating that peptide drugs can target and bind to PCSK9 protein.

[0044] Example 4

[0045] Example 4 is used to explore the increasing effect of polypeptide drugs on LDL receptors:

[0046] HepG2 cells were used as a model and cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at a temperature of 37° C. and a CO 2 concentration of 5%.

[0047] HepG2 cells were seeded in six-well plates and cultured overnight. After the cells adhered to the wall the next day, the old culture medium was discarded and replaced with fresh culture medium containing NSP or Pep2-8 (50 μM).

[0048] After the cells and materials were incubated for 4 h, 8 h, 12 h, and 24 h, the cells were lysed and the proteins were extracted for gel electrophoresis experiments. After transfer and blocking, the membrane was incubated with the primary antibody (LDLR) at 4 °C overnight, washed 3 times with TBST, incubated with the secondary antibody at room temperature for 1 h, and washed three times with TBST before color development.

[0049] The imprint results are as follows Figure 6 As shown, NSP can increase LDL receptors with better temporal persistence than Pep2-8.

[0050] Example 5

[0051] Example 5 is used to explore the effect of polypeptide drugs on increasing LDL receptors in vivo:

[0052] Six-week-old female C57BL / 6 healthy mice were divided into PBS group and NSP group. The mice were injected with 10 mg / kg of NSP via tail vein, twice a day for 3 consecutive days.

[0053] Two hours after the last administration, the mice were killed, and the livers were removed and ground with lysis buffer to extract proteins for Western Blot experiments.

[0054] The imprint results are as follows Figure 7 As shown, NSP can significantly increase the expression of LDL receptor in mouse liver.

[0055] The present invention designs, synthesizes and characterizes novel polypeptide drugs through Examples 1 to 5, and finds that NSP can specifically recognize PCSK9 protein and better bind to PCSK9 protein through multivalent bond synergy, and competitively inhibit the binding of PCSK9 protein to LDLR. NSP can increase the recovery of LDLR both in vitro and in vivo to achieve efficient inhibition of PCSK9 and downregulation of serum LDLC, and therefore can be used as a polypeptide drug for treating hypercholesterolemia.

[0056] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A polypeptide drug, characterized in that: The active ingredients of the drug are n-hexadecanoic acid, a polypeptide sequence FFVLK, PEG4 and a polypeptide sequence TVFTSWEEYLDWV which are sequentially connected through amide bonds.

2. Use of a polypeptide drug according to claim 1 in the preparation of a drug for treating hypercholesterolemia.

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