Preparation method and application of self-assembled polypeptide targeting CD36 protein

Abstract

The present invention discloses a preparation method and application of a self-assembling polypeptide targeting CD36 protein, belonging to the technical field of biomedicine. The self-assembling polypeptide is composed of an R3 specific targeting unit, an R2 self-assembling unit, and an R1 hydrophobic unit sequentially connected by amide bonds. The R1 hydrophobic unit is an alkyl carboxylic acid with C8-C20, the R2 self-assembling unit is a multiple hydrogen bond polypeptide sequence, and the R3 specific targeting unit is a polypeptide with specific targeting to CD36 protein screened from a polypeptide library. The self-assembling polypeptide of the present invention can bind to CD36. The self-assembling polypeptide of the present invention is obtained by solid-phase synthesis method, and has many advantages such as small relative molecular weight, high specificity, and low cost. It shows unique advantages in the targeted diagnosis and treatment of tumors, and has extremely important and extensive application prospects and good economic and social effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a preparation method and application of a self-assembling polypeptide that has a targeting effect on CD36 protein. Background Art

[0002] CD36 is a scavenger receptor that plays an important role in lipid metabolism and is involved in angiogenesis, inflammatory responses, atherosclerotic thrombosis, etc. In the immune system, CD36 mediates the uptake of fatty acids by tumor-infiltrating CD8+ T cells, causing lipid peroxidation and ferroptosis, resulting in reduced production of tumor toxic factors and T cell exhaustion. At the same time, studies have shown that CD36 is a specific target of Treg cells, and CD36 is only highly expressed in Treg cells in the tumor microenvironment. Most importantly, targeting CD36 on Treg cells does not cause autoimmune reactions, but significantly enhances the anti-tumor activity of immune cells. CD36 antibodies exhibit anti-tumor activity in mouse tumor models and have a synergistic effect with PD-1 antibodies. Therefore, specifically blocking CD36 and hindering the recognition and transmembrane transport of fatty acids is an effective way to precisely regulate the fatty acid metabolism of Treg cells and enhance the function of immune cells.

[0003] Through previous literature research, we found that there are few reports on polypeptide drugs targeting CD36 for immunotherapy. Therefore, continuing to provide preparations with considerable CD36 affinity and providing the application of such molecules in diagnosing and treating diseases remains an important issue in this field. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a self-assembling polypeptide that has a targeting effect on CD36 protein.

[0005] To achieve the above purpose, the present invention provides the following technical solution:

[0006] A self-assembling polypeptide that has a targeting effect on CD36 protein, wherein the self-assembling polypeptide is composed of an R3 specific targeting unit, an R2 self-assembling unit, and an R1 hydrophobic unit that are sequentially connected by amide bonds; the R1 hydrophobic unit is an alkyl carboxylic acid with C8-C20, the R2 self-assembling unit is a multiple hydrogen bond polypeptide sequence, and the R3 specific targeting unit is a polypeptide that has specific targeting to CD36 protein and is screened from a polypeptide library.

[0007] Further, the starting acid of the R1 hydrophobic unit is an alkyl carboxylic acid with 8 to 20 carbon atoms; the starting acid of the R3 specific targeting unit is an amino acid immobilized on Wang resin; the common starting acids of the R2 self-assembling unit and the R3 specific targeting unit are amino acids protected by Fmoc or Boc; the amino acid sequence of the self-assembling polypeptide is: GViRTIR, GGGViRTIR, GVIRTIR, GGGVIRTIR.

[0008] Further, a polypeptide derivative in the self-assembling polypeptide molecule that has one or more amino acids deleted, inserted, or replaced and has the same biological function as the self-assembling polypeptide molecule.

[0009] Further, the polypeptide has a targeting effect on CD36 and can specifically bind to CD36.

[0010] Further, a method for preparing a self-assembling polypeptide having a targeting effect on CD36 protein, characterized in that: an amino acid immobilized on Wang resin, an amino acid protected by Fmoc or Boc, and an alkyl carboxylic acid with 8 to 20 carbon atoms are used as raw materials, and the self-assembling polypeptide is prepared by a solid-phase synthesis method;

[0011] The synthesis sequence of the self-assembling polypeptide is that R3, R2, and R1 are sequentially connected via amide bonds, and each amino acid is deprotected and then condensed in sequence, repeating multiple times until all amino acids and alkyl carboxylic acids are connected.

[0012] Further, an application of a self-assembling polypeptide having a targeting effect on CD36 protein in the preparation of an anti-tumor drug.

[0013] Further, the anti-tumor drug contains the polypeptide and a drug active ingredient, or contains the polypeptide and a drug delivery carrier.

[0014] Further, the anti-tumor drug is any pharmaceutically acceptable dosage form.

[0015] Further, the preferred dosage form of the anti-tumor drug is an injection preparation.

[0016] Further, the anti-tumor drug is any pharmaceutically acceptable dose.

[0017] The beneficial effects of the present invention are as follows:

[0018] 1. The self-assembling polypeptide provided by the present invention has a targeting effect on CD36, can specifically bind to CD36 and achieve the function of blocking. In the present invention, after the polypeptide recognizes the CD36 protein, it promotes the spontaneous formation of a complex, ordered, regular, and high-level structure nanofiber with a blocking function among polypeptide molecules through non-covalent bonds or intermolecular forces (hydrogen bonds, hydrophobic bonds, Π-Π stacking, ionic bonds, van der Waals forces, electrostatic attraction, etc.), thereby blocking the function of the CD36 receptor.

[0019] 1. The self-assembling polypeptide provided by the present invention has many advantages such as small relative molecular weight, high specificity, and low cost. It shows unique advantages in the targeted diagnosis and treatment of tumors, has extremely important and broad application prospects, and also has good economic and social effects.

[0020] Other advantages, objectives, and features of the present invention will be described in the subsequent specification, and to some extent, they are obvious to those skilled in the art, or those skilled in the art can obtain teachings from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 1 It is the mass spectrometry characterization diagram of the self-assembling polypeptide in Example 1 of the invention;

[0023] Figure 2 It is the morphological feature diagram of the nano-solution under a transmission electron microscope in Example 2 of the invention;

[0024] Figure 3 It is the cell fatty acid detection diagram of the nano-solution in Example 3 of the invention;

[0025] Figure 4 It is the diagram of the change of Treg cells in the mouse tumor in Example 4 of the invention.

[0026] Figure 5 It is the inhibition result diagram of the inhibition of tumor growth by SRP in Example 4 of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following is only the preferred embodiment of the present invention and does not limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0028] The object of the present invention is to provide a self-assembling polypeptide that targets CD36 protein, meeting the need for continuously providing a substance with CD36 affinity, specifically binding to CD36 protein, and being applicable to therapeutic and diagnostic applications.

[0029] Another object of the present invention is to provide a CD36 protein-binding polypeptide, which can be used as a part of a fusion polypeptide.

[0030] To achieve the above object, the present invention adopts the following technical means:

[0031] The present invention relates to a self-assembling polypeptide that targets CD36 protein. The polypeptide comprises an R1 hydrophobic unit, an R2 self-assembling unit, and an R3 specific targeting unit, and each unit is connected via an amide bond. The structural example is as follows:

[0032] R1-R2-R3

[0033] After the R3 specific targeting unit recognizes the CD36 protein, it can bind and undergo ligand-receptor interaction, causing the R2 unit with multiple hydrogen bonds to deform, realizing the transformation from nanoparticles to nanofibers, and then covering the entire CD36 molecule, thereby affecting the functional operation of CD36.

[0034] The present invention also provides a preparation method for the self-assembling polypeptide that targets CD36 protein. The specific method is as follows:

[0035] Using amino acids immobilized on Wang resin, Fmoc- or Boc-protected amino acids, and C8-C20 alkyl carboxylic acids as raw materials, it is prepared by solid-phase synthesis.

[0036] The synthesis sequence is from R3 to R2 and then to R1. Each amino acid is deprotected and condensed in sequence, and this process is repeated until all amino acids and alkyl carboxylic acids are connected.

[0037] Example 1

[0038] A self-assembling polypeptide that targets CD36 protein (hereinafter referred to as SRP). The chemical structural formula of SRP is:

[0039] CH3(CH2)CO-FFKLV-GViRTIR

[0040] SRP is obtained by solid-phase synthesis and characterized by mass spectrometry. The results are as Figure 1 shown. The main peak of the mass spectrum is basically consistent with the molecular weight of the SRP polypeptide. Therefore, it can be considered that the target polypeptide has been successfully obtained.

[0041] Example 2

[0042] To investigate whether self-assembled polypeptides can recognize CD36 protein and undergo deformation.

[0043] Dissolve the SRP in Example 1 in DMSO, and take out 10 μL from it and dissolve it in 990 μL of pure water to prepare a nano-solution of SRP.

[0044] Co-incubate the above nano-solution with CD36 protein at a concentration of 1×10-7 M, and observe the morphological characteristics at 0 h, 1 h, 4 h, and 6 h under a transmission electron microscope. The results are as Figure 2 shown. SRP can recognize CD36 well and undergo deformation.

[0045] Example 3

[0046] To investigate whether self-assembled polypeptides can affect the lipid uptake function of CD36 overexpressing cells.

[0047] Prepare a nano-solution of SRP at a concentration of 50 μM in Example 1 and co-incubate it with CD36 overexpressing cells Raw264.7 for 12 h, then incubate with Bodipy-C12 for 30 min, and detect the fatty acid uptake of cells by flow cytometry. The results are as Figure 3 shown. SRP can inhibit the lipid uptake function of CD36 overexpressing cells well.

[0048] Example 4

[0049] To investigate whether self-assembled polypeptides can affect CD36 overexpressing Treg cells in the tumor immune microenvironment and bring about tumor treatment effects.

[0050] Use B16-F10 cells to establish a tumor model in C57BL / 6J mice, observe the tumor growth situation, and start injecting SRP polypeptide solution at a concentration of 7.5 mg / Kg on the 8th day after tumor cell injection. After five treatments, use flow cytometry to check the changes of Treg cells in the tumor tissue. The results are as Figure 4 shown. The SRP polypeptide can reduce the number of Treg cells at the tumor tissue site. Observe the tumor growth status, and the results are as Figure 5 shown. The SRP polypeptide can also inhibit the growth of tumors to a certain extent.

[0051] 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 them. 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 self-assembling polypeptide with a targeting effect on CD36 protein, wherein the chemical structural formula of the self-assembling polypeptide is: CH3(CH2)CO-FFKLV-GViRTIR.

2. A self-assembling polypeptide having a targeting effect on CD36 protein according to claim 1, characterized in that: The polypeptide has a targeting effect on CD36 and can specifically bind to CD36.

3. Use of a self-assembling polypeptide having a targeting effect on CD36 protein according to any one of claims 1-2 in the preparation of a drug for anti-melanoma tumors.

4. The use according to claim 3, characterized in that: The anti-melanoma drug comprises the polypeptide and a pharmaceutically active ingredient, or comprises the polypeptide and a drug delivery carrier.

5. The use according to claim 3, characterized in that: The anti-melanoma drug can be in any therapeutically acceptable dosage form.

6. The use according to claim 3, characterized in that: The dosage form of the anti-melanoma drug is an injectable preparation.

7. The use according to claim 3, characterized in that: The anti-melanoma drug can be any drug in a therapeutically acceptable dose.

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