A polypeptide and use thereof in the preparation of a medicament for protecting the blood-brain barrier

Abstract

The application discloses a polypeptide and application thereof in preparation of a medicine with blood-brain barrier protection function. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1, and is specifically VKKEAEKDKYLE. The TAT-QFNP12 polypeptide is synthesized according to the application, and the TAT-QFNP12 polypeptide can target and inhibit the expression of MMP-9 in the brain, so as to achieve the purpose of reducing pathological damages such as blood-brain barrier destruction and secondary brain edema of brain injury diseases.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a polypeptide and its use in the preparation of drugs with blood-brain barrier protection function. Background Technology

[0002] Blood-brain barrier (BBB) ​​damage is a significant cause of secondary cerebral edema, neuronal cell damage, and neuroinflammation following neurological diseases such as stroke, traumatic brain injury, and encephalitis, leading to poor patient prognosis. Matrix metalloproteinase 9 (MMP-9) exerts a detrimental effect on the BBB by degrading the neurovascular matrix. MMP-9 is the main type of MMPs that degrades the BBB matrix and cell junction components; therefore, inhibiting MMP-9 production or activity is a crucial strategy for protecting the BBB. However, current MMP-9 inhibitors cannot inhibit MMP-9 expression in specific cell types and have failed in clinical trials for neurological diseases due to significant side effects. Therefore, developing methods to specifically inhibit intracerebral MMP-9 production, targeting the key cellular and molecular mechanisms of MMP-9 production after acute brain injury, to protect the BBB and mitigate brain damage is an important direction for achieving neuroprotection and improving prognosis.

[0003] Previous studies have clearly shown that the transcriptional expression of MMP-9 is regulated by the transcription factors Smad2 / 3 (Smad2 / 3). Smad2 / 3 is a receptor-regulated transcription factor, an intracellular signal transducer and transcriptional regulator activated by TGFβ (transforming growth factor β) and activin type 1 receptor kinase. It activates the transcriptional expression of target genes by binding to TRE elements in the promoter regions of many TGFβ-regulated genes and forming a complex with SMAD4. MMP-9 has been confirmed as an important target gene of Smad2 / 3 signaling. How to inhibit MMP-9 expression through the transcriptional pathway to alleviate the pathological changes caused by elevated MMP-9 levels has become a challenge for MMP-9 intervention. Traditional methods for inhibiting Smad2 / 3 use small molecule inhibitors of the TGFβ receptor (TGFβR), but these have significant limitations in the brain. Several clinical trials attempting to intervene in brain pathological damage such as blood-brain barrier disruption have failed. Further research is needed to explore more effective intervention methods to inhibit the Smad2 / 3-MMP9 pathway.

[0004] Smad2 / 3's transcriptional regulation of downstream target genes depends on its phosphorylation and nuclear translocation to exert transcriptional activity. When transcription ends, phosphorylated Smad2 / 3 needs to be dephosphorylated to exit the nucleus and terminate transcription. Research has found that Smad2 / 3 dephosphorylation depends on the dephosphorylation of the protein phosphatase PPM1A. PPM1A is mainly enriched in the cytoplasm under physiological conditions. Whether, after exposure to damaging factors, enhancing PPM1A nuclear translocation can promote Smad2 / 3 dephosphorylation, thereby promoting Smad2 / 3 nuclear translocation to terminate MMP-9 transcriptional expression, represents an important innovative approach for targeted intervention in MMP-9 transcriptional expression. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a polypeptide and its use in the preparation of a drug with blood-brain barrier protection function. The polypeptide drug prepared by this invention can protect the blood-brain barrier and effectively treat symptoms such as secondary cerebral edema, nerve cell damage, and neuroinflammation caused by neurological diseases such as stroke, craniocerebral trauma, and encephalitis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A polypeptide having the amino acid sequence shown in SEQ ID NO.1, specifically VKKEAEKDKYLE.

[0008] Furthermore, the polypeptide also includes a polypeptide sequence that has more than 80% homology with the sequence shown in SEQ ID NO.1 and has the same function.

[0009] Furthermore, the polypeptide also includes a membrane-penetrating peptide bound to its N-terminus.

[0010] Furthermore, the membrane-penetrating peptide is TAT, and its amino acid sequence is YGRKKRRQRRR.

[0011] An MMP-9 inhibitor comprising the aforementioned peptide.

[0012] Furthermore, the inhibitor suppresses MMP-9 transcriptional expression by blocking the binding of NDRG2 to PPM1A.

[0013] A drug with blood-brain barrier protection, comprising the aforementioned peptide, or an MMP-9 inhibitor, and pharmaceutically acceptable excipients thereof.

[0014] A medicine for treating a neurological disorder with a poor prognosis, comprising the aforementioned peptide, or an MMP-9 inhibitor, and pharmaceutically acceptable excipients thereof.

[0015] Furthermore, neurological disorders include stroke, traumatic brain injury, or encephalitis.

[0016] The beneficial effects of this invention are:

[0017] This invention synthesizes the TAT-QFNP12 polypeptide, which can block the binding of NDRG2 / PPM1A and inhibit the expression of MMP-9, thereby reducing the pathological damage such as blood-brain barrier disruption and secondary cerebral edema in brain injury diseases.

[0018] This invention provides a novel target for inhibiting MMP-9 in the brain. The initially developed peptide drug has the characteristics of high brain enrichment, strong MMP-9 inhibition, few peripheral side effects, and strong neuroprotective effect, and has the potential for clinical translational application. Attached Figure Description

[0019] Figure 1 Blood-brain barrier permeability analysis of TAT-QFNP12 peptide;

[0020] Figure 2 Analysis of cell membrane permeability of TAT-QFNP12 peptide;

[0021] Figure 3 The effect of TAT-QFNP12 peptide on the NDRG2 / PPM1A-Smad2 / 3-MMP9 signaling pathway;

[0022] Figure 4 The effect of TAT-QFNP12 peptide on reducing blood-brain barrier disruption and cerebral edema after cerebral hemorrhage;

[0023] Figure 5 The effects of TAT-QFNP12 polypeptide on liver and kidney function in animals;

[0024] Figure 6 HE-stained images show the biosafety of the TAT-QFNP12 peptide in the cellular morphology of mouse heart, lung, liver, kidney, and brain. Detailed Implementation

[0025] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0026] Example 1: Design and Synthesis of Peptides

[0027] Using computer-simulated molecular docking technology, the protein-binding domains of NDRG2 / PPM1A were preliminarily analyzed. It was found that the 111-114 and 221-228 domains of NDRG2 can bind to the 364-366 and 221-228 domains of PPM1A, respectively.

[0028] Further molecular biological studies, including protein truncation and immunoprecipitation, clarified that the 221-228 domains of NDRG2 and the 302-313 domains of PPM1A play the most crucial roles in the protein-protein binding of NDRG2 / PPM1A. These domains can serve as key components in the development of NDRG2 / PPM1A signaling inhibitors. Since PPM1A is an important intracellular enzyme, broad inhibition of PPM1A may have significant side effects. Therefore, designing inhibitors targeting the 221-228 domains of the NDRG2 protein to block NDRG2 / PPM1A binding is a safer and more effective approach.

[0029] Based on this, a polypeptide consisting of 12 amino acids was designed for this domain and synthesized by Sangon Biotech (Shanghai) Co., Ltd., with the sequence VKKEAEKDKYLE (SEQ ID NO.1).

[0030] Meanwhile, to enhance the ability of this polypeptide to cross the blood-brain barrier and penetrate cell membranes to enter brain cells, a membrane-penetrating peptide TAT (YGRKKRRQRRR) was attached to the N-terminus of the polypeptide, ultimately forming a 23-amino acid biopeptide with blood-brain barrier penetration capability and targeting of NDRG2 / PPM1A signaling in the brain. This biopeptide was named TAT-QFNP12, and its sequence is: YGRKKRRQRRR-VKKEAEKDKYLE (SEQ ID NO.2).

[0031] Example 2: The effect of TAT-QFNP12 peptide

[0032] 1. Brain penetration of TAT-QFNP12 peptide

[0033] (1) Using FITC-labeled TAT-QFNP12 peptide, the peptide fluorescence signal in the cerebral cortex and hippocampus was detected at 1 h and 6 h after tail vein injection in mice. A significant green fluorescence signal of the peptide could be detected at 1 h and could be stably maintained for more than 6 h. The control group was given an equal volume of physiological saline via tail vein injection.

[0034] (2) When astrocytes were treated with FITC-labeled TAT-QFNP12 peptide for 1 h, green fluorescence signal could be detected in the cells, and the intensity of the fluorescence signal increased continuously with the increase of treatment concentration.

[0035] like Figure 1 and 2 As shown, animal and cell experiments have verified that TAT-QGNF12 has strong brain permeability.

[0036] 2. Effects of TAT-QFNP12 peptide on the NDRG2 / PPM1A-MMP9 signaling pathway

[0037] The inhibitory effect of the peptide on the binding of NDRG2 / PPM1A protein in cells was analyzed by immunoprecipitation, and the inhibitory effect of the peptide on the expression of MMP-9 and the phosphorylation level of Smad2 / 3 in the brain after cerebral hemorrhage was analyzed by immunoblotting.

[0038] like Figure 3 As shown, Figure 3 The values ​​of A and a indicate that the TAT-QFNP12 peptide has the ability to block the binding of NDRG2 / PPM1A. Figure 3 Figures B and b indicate that the TAT-QFNP12 peptide inhibits Smad2 / 3 phosphorylation activation and MMP-9 expression, suggesting that the TAT-QFNP12 peptide can inhibit the NDRG2 / PPM1A-Smad2 / 3-MMP9 signaling pathway.

[0039] 3. The protective effect of TAT-QFNP12 peptide on the blood-brain barrier

[0040] The level of Evans blue dye entering the brain parenchyma through the blood-brain barrier was analyzed by tail vein injection, and the degree of blood-brain barrier disruption and cerebral edema were evaluated. The dosage of the peptide was 20 mg / kg.

[0041] like Figure 4 As shown, Figure 4 The results from A and a indicate that the TAT-QFNP12 peptide can effectively treat blood-brain barrier damage and has a blood-brain barrier protective function. Figure 4 The result from B indicates that the TAT-QFNP12 polypeptide can alleviate cerebral edema and has a neuroprotective effect in reducing cerebral edema.

[0042] 4. Biocompatibility of TAT-QFNP12 peptide

[0043] (1) Effects on liver and kidney function in animals

[0044] Normal mice were injected with TAT-QFNP12 intravenously for 7 consecutive days at concentrations of 20 mg / kg and 100 mg / kg. The levels of important liver and kidney function biomarkers in plasma, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), direct bilirubin (DBIL), blood urea nitrogen (BUN), and creatinine (CRE), were then measured. The results are as follows: Figure 5 As shown, Figure 5 In the diagram, P20 represents the 20 mg / kg TAT-QFNP12 treatment group; P100 represents the 100 mg / kg TAT-QFNP12 treatment group.

[0045] like Figure 5 As shown, after continuous injection of different concentrations of TAT-QFNP12 peptide for 7 days, it had no significant effect on the liver and kidney function of mice.

[0046] (2) Effects on the cellular morphology of mouse heart, lung, liver, kidney and brain

[0047] TAT-QFNP12 peptide was intravenously injected into mice daily at concentrations of 20 mg / kg and 100 mg / kg, with an equal volume of physiological saline as a control group, for 7 consecutive days. The cytological toxicity of the peptide on the heart, lungs, liver, kidneys and brain of mice was then examined.

[0048] like Figure 6 As shown, after continuous injection of different concentrations of TAT-QFNP12 peptide for 7 days, it had no significant toxic effects on the cellular morphology of the heart, lungs, liver, kidneys and brain of mice.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

2.

2. A drug with blood-brain barrier protection function, characterized in that, The drug comprises the polypeptide of claim 1, and pharmaceutically acceptable excipients thereof.

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