A BNCT boron drug targeting thrombus fibrin and a screening method thereof

Abstract

The present application relates to a kind of BNCT boron medicine targeting thrombus fibrin and its screening method, it is characterized in that, using the structural similarity of BPA and aromatic amino acid carries out BNCT boron medicine design, first screening out the targeting polypeptide of recognition thrombus fibrin, then through BPA replacement and modification obtain the thrombus fibrin targeting peptide containing BPA, finally through iterative screening method obtains the BNCT boron medicine that can be used for BNCT technology by neutron bombardment realizes thrombus directional clearance.

Description

Technical Field

[0001] This invention belongs to the field of boron neutron capture therapy (BNCT), specifically relating to a BNCT boron drug targeting thrombus fibrin and its screening method, especially a boron drug for BNCT synthesized using polypeptide solid-phase synthesis technology and its screening method. Background Technology

[0002] Boron neutron capture therapy (BNCT) is a treatment that utilizes... 10 The high-tech method of boron atoms absorbing thermal neutrons and exploding inside tumor cells to selectively eliminate tumor cells within a single cell range involves the core work of [the process of] [destruction of tumor cells containing boron atoms]. 10 Boron-based drugs are specifically delivered to tumor cells, and each tumor cell contains at least 10 boron atoms. 9 indivual 10 The boron atom must be present, and the concentration of boron in tumor cells must be more than three times that of the surrounding normal cells.

[0003] Currently, the FDA has approved two BNCT drugs: BSH (thiol polyhedral boron cage compound) and BPA (4-boron-L-phenylalanine), with BPA being more widely used. Various boron-loaded targeted drugs, such as low molecular weight boron compounds, boron-doped peptides, antibody fragments and various proteins, intact antibodies and antibody-based conjugates, and liposomes, fail to meet the therapeutic requirements of BNCT due to problems such as low drug loading, poor penetration, and high systemic toxicity. Ultimately, this stems from the poor targeting and low boron loading of these boron drugs.

[0004] A thrombus is a small mass that forms on the surface of a blood vessel in the cardiovascular system at a site of rupture or repair. It is composed of fibrin, deposited platelets, and accumulated white blood cells and red blood cells. Because thrombosis obstructs blood flow, it can cause ischemic symptoms in the corresponding organs, as well as serious symptoms such as limb necrosis, and in severe cases, it can threaten the patient's life. After endothelial injury, the body initiates intrinsic and extrinsic coagulation mechanisms. Fibrinogen is cleaved into a fibrin cross-linked network under the catalysis of thrombin, trapping white blood cells, red blood cells, etc., in the blood and forming a thrombus with platelets. Fibrin accounts for 50%-60% of the dry weight of the thrombus, and the cross-linked structures form a network structure, forming the thrombus skeleton. Fibrin is abundant in thrombi, but its content in the blood, blood vessel walls, and tissues and organs is very low.

[0005] Thromboplastin is a structural protein containing serine and aspartic acid residues. It is one of the earliest discovered clotting factors in humans, appearing as an elongated ellipsoid. It is a dimer composed of three pairs of polypeptide chains (one α-chain, one β-chain, and one γ-chain) linked by disulfide bonds, and primarily functions in blood coagulation and hemostasis. Utilizing the fact that fibrin accounts for 50%-60% of the dry weight of thrombi and forms a cross-linked network structure, targeting fibrin offers an excellent opportunity for thrombus non-component thrombus (BNCT) therapy. However, currently, there is a lack of boron drugs that can effectively target thrombofibrin in BNCT; only BPA and BSH, which lack thrombofibrin targeting, exist on the market. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a boron drug for BNCT that targets thrombofibrin and a screening method thereof, particularly screening out boron drugs that can be synthesized using polypeptide solid-phase synthesis technology, target thrombofibrin, and can be used for BNCT.

[0007] Currently, BNCT drugs are mainly based on BPA (4-boron-L-phenylalanine). This invention designs boron drugs for BNCT based on the structural similarity between BPA and aromatic amino acids, especially tyrosine, screens out tyrosine-containing targeted peptides that target thrombofibrin, and further completes BPA substitution and sequence optimization, as well as solid-phase synthesis of thrombofibrin-targeting peptides.

[0008] To achieve this objective, the present invention employs the following technical solution.

[0009] A boron drug targeting thrombus fibrin and its screening method, characterized by comprising the following steps: Based on the composition and structural characteristics of thrombi, S1 uses methods such as molecular dynamics simulation to screen out targeted peptides that can specifically bind to thrombus fibrin. S2 screens for target peptides containing aromatic amino acids from thrombofibrin-targeting peptides, especially target peptides with aromatic amino acids at the amino and / or carboxyl termini. S3 utilizes the structural similarity between BPA and aromatic amino acids to design BNCT boron drugs, and uses the target peptides after replacing aromatic amino acids with BPA to dock with thrombus fibrin, screening out thrombus fibrin targeting peptides with BPA. S4 adds BPA to the thrombofibrin targeting peptide with BPA, further screens and optimizes it, and optimizes the affinity and specificity of the thrombofibrin targeting peptide through iterative design, finally obtaining a thrombofibrin specific targeting peptide containing multiple BPAs. S5 utilizes solid-phase polypeptide synthesis technology to prepare thrombofibrin-specific targeting peptides containing multiple BPAs.

[0010] Furthermore, by utilizing the structural similarity between BPA and aromatic amino acids, especially tyrosine, boron drugs for BNCT were designed. After screening for targeting peptides containing aromatic amino acids that target thrombofibrin, BPA substitution and sequence optimization were performed.

[0011] Furthermore, taking advantage of the consistency between BPA and amino acid structures, BNCT boron drugs targeting thrombofibrin were synthesized using peptide solid-phase synthesis technology.

[0012] Furthermore, the thrombofibrin-specific targeting peptide sequence containing BPA was obtained through screening and found to be: (amino terminus) BPA-ESDVSAQME-G-BPA (carboxyl terminus).

[0013] Furthermore, the thrombofibrin-specific targeting peptide sequence containing BPA was obtained through screening: (amino terminus) BPA-SSELEKHQL-BPA (carboxyl terminus).

[0014] Furthermore, the application of a boron drug targeting thrombofibrin in BNCT and its screening method in the field of BNCT. Attached Figure Description Figure 1 The binding structure of the targeting peptide BPA-ESDVSAQME-G-BPA to thrombofibrin 3GHG. Figure 2 The binding structure of the targeting peptide BPA-SSELEKHQL-BPA to thrombofibrin 3GHG. Figure 1 and Figure 2 In the diagram, 1-1 represents the crystal structure of thrombofibrin, 1-2 represents the targeting polypeptide, 2-1 represents the amino acid residues of thrombofibrin that directly interact with the targeting polypeptide, 2-2 represents the targeting polypeptide, and 3-2 represents the sequence of the targeting polypeptide.

[0015] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0016] The boron drug for BNCT targeting thrombus fibrin and its screening method provided by this invention can load a targeting system onto BPA, which currently lacks thrombus fibrin targeting function, to achieve targeted enrichment of BPA towards thrombus fibrin. This enables BNCT treatment for thrombi, allowing thrombus patients undergoing conservative treatment and home rehabilitation to receive effective BNCT therapy. The thrombus in the blood vessel is effectively removed by boron drug combined with neutron bombardment, without the need for surgery. A single treatment course can complete the targeted removal of thrombi in less than an hour. In particular, for patients with multiple thrombi in the blood vessel, all thrombi can be removed in one session, reducing the mobility impairment caused by the disease and the huge economic burden of accompanying care. Detailed Implementation

[0017] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0018] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products. Example 1

[0019] (1) Based on the crystal structure of thrombofibrin 3GHG, the structure of thrombofibrin 3GHG was analyzed using the corresponding bioinformatics software.

[0020] (2) Using bioinformatics tools and big data databases, we screened for specific binding target peptides of fibrinogen 3GHG. There are multiple target peptide recognition sites on fibrinogen 3GHG, such as Arg-Gly-Asp (RGD) sequence, Arg-Ala-Asp-Val (RADV) and Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence, etc. In particular, target peptides with aromatic amino acids at the amino terminus and / or carboxyl terminus were screened. The target peptide of fibrinogen 3GHG was obtained: (amino terminus) ESDVSAQMEY (carboxyl terminus).

[0021] (3) Taking advantage of the structural similarity between BPA and aromatic amino acids, BNCT boron drugs were screened. Using software tools, aromatic amino acids in the target peptide of thrombofibrin 3GHG were replaced with BPA. The recognition and binding characteristics of the peptide after BPA replacement of aromatic amino acids with thrombofibrin 3GHG were analyzed and screened.

[0022] (4) Optimize the target peptide based on peptide-protein interaction calculations, including molecular docking method and molecular dynamics dynamic screening method. Based on the determination of the thrombofibrin 3GHG target peptide containing aromatic amino acids, optimize the affinity and specificity of the target peptide through iterative design and screening.

[0023] (5) BPA was added to the amino and carboxyl ends of the screened thrombofibrin 3GHG targeting peptides that already contained BPA, and further screening and optimization were carried out. The affinity and specificity of the thrombofibrin 3GHG targeting peptides were optimized through iterative design, and finally, a thrombofibrin 3GHG specific targeting peptide containing multiple BPAs was obtained: (amino end) BPA-ESDVSAQME-G-BPA (carboxyl end). The screening results are summarized in Table 1 below, where the lower the calculated score, the higher the binding force between the targeting peptide and the receptor protein. Table 1. Summary of screening results based on the thrombofibrin 3GHG-targeting peptide ESDVSAQMEY

[0024] (6) Using solid-phase polypeptide synthesis technology, a thrombofibrin 3GHG-specific targeting peptide containing multiple BPAs was prepared: (amino terminus) BPA-ESDVSAQME-G-BPA (carboxyl terminus), and used for the development and application of boron drugs related to BNCT treatment. Example 2

[0025] (1) Based on the crystal structure of thrombofibrin 3GHG, the structure of thrombofibrin 3GHG was analyzed using the corresponding bioinformatics software.

[0026] (2) Using bioinformatics tools and big data databases, we screened for specific binding target peptides of fibrinogen 3GHG. There are multiple target peptide recognition sites on fibrinogen 3GHG, such as the Arg-Gly-Asp (RGD) sequence, Arg-Ala-Asp-Val (RADV) and Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence. In particular, we screened for target peptides with aromatic amino acids at the amino terminus and / or carboxyl terminus. The target peptide of fibrinogen 3GHG was obtained as: (amino terminus) SSELEKHQLY (carboxyl terminus).

[0027] (3) Taking advantage of the structural similarity between BPA and aromatic amino acids, BNCT boron drugs were screened. Using software tools, aromatic amino acids in the target peptide of thrombofibrin 3GHG were replaced with BPA. The recognition and binding characteristics of the peptide after BPA replacement of aromatic amino acids with thrombofibrin 3GHG were analyzed and screened.

[0028] (4) Optimize the target peptide based on peptide-protein interaction calculations, including molecular docking method and molecular dynamics dynamic screening method. Based on the determination of the thrombofibrin 3GHG target peptide containing aromatic amino acids, optimize the affinity and specificity of the target peptide through iterative design and screening.

[0029] (5) BPA was added to the amino and carboxyl ends of the screened thrombofibrin 3GHG targeting peptides containing BPA, and further screening and optimization were carried out. The affinity and specificity of the thrombofibrin 3GHG targeting peptides were optimized through iterative design. Finally, a thrombofibrin 3GHG specific targeting peptide containing multiple BPAs was obtained: (amino end) BPA-SSELEKHQL-BPA (carboxyl end). The screening results are summarized in Table 2 below. The lower the calculated score, the higher the binding force between the targeting peptide and the receptor protein. Table 2 Summary of screening results based on the thrombofibrin 3GHG-targeting peptide SSELEKHQLY

[0030] (6) Using solid-phase polypeptide synthesis technology, a thrombofibrin 3GHG-specific targeting peptide containing multiple BPAs was prepared: (amino terminus) BPA-SSELEKHQL-BPA (carboxyl terminus), and used for the development and application of boron drugs related to BNCT treatment.

[0031] Based on the above description of the present invention, and in accordance with common technical knowledge and conventional methods in the field, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention without departing from the basic technical concept of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for screening BNCT boron drugs targeting thrombofibrin, characterized in that, Includes the following steps: Based on the composition and structural characteristics of thrombi, S1 uses molecular dynamics simulation to screen for targeted peptides that can specifically bind to thrombus fibrin. S2 screens for target peptides containing aromatic amino acids from the target peptides of thrombofibrin, wherein the target peptides are target peptides with aromatic amino acids at the amino terminus and / or carboxyl terminus. S3 utilizes the structural similarity between BPA and aromatic amino acids to design BNCT boron drugs, and uses the target peptides after replacing aromatic amino acids with BPA to dock with thrombus fibrin, screening out thrombus fibrin-targeting peptides with BPA. S4 adds BPA to the thrombofibrin-targeting peptide with BPA, further screens and optimizes it, and optimizes the affinity and specificity of the thrombofibrin-targeting peptide through iterative design, finally obtaining a thrombofibrin-specific targeting peptide containing 2-3 BPAs. S5 utilizes solid-phase peptide synthesis technology to prepare thrombofibrin-specific targeting peptides containing 2-3 BPAs.

2. The method for screening BNCT boron drugs targeting thrombofibrin according to claim 1, characterized in that, Boron drugs for BNCT were designed by utilizing the structural similarity between BPA and aromatic amino acids. After screening for targeted peptides containing aromatic amino acids that target thrombofibrin, BPA substitution and sequence optimization were performed, wherein the aromatic amino acid is tyrosine.

3. The method for screening BNCT boron drugs targeting thrombofibrin according to claim 1, characterized in that, Taking advantage of the structural similarity between BPA and amino acids, boron drugs targeting thrombofibrin were synthesized using peptide solid-phase synthesis technology.

4. A BNCT boron drug targeting thrombofibrin, characterized in that, The BNCT boron drug targeting thrombofibrin was obtained by screening using the screening method described in claim 1. The sequence of the thrombofibrin-specific targeting polypeptide in the BNCT boron drug from the amino terminus to the carboxyl terminus is: X-SEQ ID NO:1-GX, where X is BPA.

5. A BNCT boron drug targeting thrombofibrin, characterized in that, The BNCT boron drug targeting thrombofibrin was obtained by screening using the screening method described in claim 1. The sequence of the thrombofibrin-specific targeting polypeptide in the BNCT boron drug from the amino terminus to the carboxyl terminus is: X-SEQ ID NO:2-X, where X is BPA.

6. The application of the screening method for BNCT boron drugs targeting thrombofibrin as described in any one of claims 1-3 in the preparation of BNCT boron drugs.

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