CN104628822B - The specific antagonist peptide and its derivative of a kind of Advanced Glycation End Product Receptors and application - Google Patents

Abstract

The invention belongs to biomedicine field, and in particular to the specific antagonist peptide and its derivative of a kind of Advanced Glycation End Product Receptors and application.The amino acid residue sequence of the specific antagonist peptide of described Advanced Glycation End Product Receptors is：Ala‑Pro‑Asp‑Thr‑Lys‑Thr‑Gln.Its derivative is on the specific antagonist peptide ammino acid side-chain radical, the aminoterminal of the specific antagonist fragments of peptides or c-terminus carry out routinely modifying obtained product, or be that the product obtained by the label of polypeptide or Protein Detection or purifying is connected on the specific antagonist peptide.The specific antagonist peptide and its derivative of described Advanced Glycation End Product Receptors can combine in vivo and specifically RAGE in vitro, treat nerve degenerative diseases by the effect of antagonism of RAGE, it can also be used to Study on Correlative Mechanisms.Achievement of the present invention can be widely used in Med Biol field, produce huge social and economic effects.

Description

A specific antagonistic peptide for receptors of advanced glycation end products and its derivatives and application

technical field

The invention belongs to the field of biomedicine, and specifically relates to a specific antagonistic peptide for receptors of advanced glycation end products, derivatives and applications thereof.

Background technique

Receptor for advanced glycation end products (RAGE) is a multi-ligand signal transduction receptor, and its ligands include advanced glycation end products (AGE), β-amyloid protein (Aβ )Wait. RAGE mediates the binding of ligands such as AGE and Aβ on the cell surface, activates a variety of signal transduction mechanisms, and leads to oxidative stress and abnormal cell function. In neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease It plays a pivotal role in the pathogenesis of diseases such as PD.

Alzheimer's disease (AD), also known as senile dementia, is a central nervous system lesion that occurs in the early stage of old age and is characterized by progressive cognitive dysfunction and behavioral impairment. It is a chronic progressive central nervous system lesion. Dementia is the most common type. Clinical manifestations include progressive memory impairment, aphasia, apraxia, agnosia, loss of visuospatial ability, loss of abstract thinking and calculation ability, personality and behavior changes, according to the annual report released by Alzheimer's Disease International, the number of dementia in the world in 2010 was 35.6 million , will reach 65.7 million in 2030, and there may be 115.4 million people in 2050, but so far there is no effective treatment for AD. It has an extremely far-reaching impact and an immeasurable contribution to society.

The pathogenesis of Alzheimer's disease (AD) is mainly β-amyloid protein (β-amyloid protein, Aβ) deposits in the brain to form senile plaques, resulting in nerve cell apoptosis and inflammatory pathology caused by microglia Changes (Ellis et al, 1996) More and more evidence shows that Aβ in the peripheral blood of AD patients damages vascular endothelial cells, destroys the blood-brain barrier, changes the permeability of the blood-brain barrier, and further increases the passage of peripheral blood Aβ through the blood-brain barrier , deposited in the brain, aggravating the pathological symptoms of AD (Biron K E et al, PIOS ONE 20116(8):e23789). RAGE is an important carrier involved in Aβ transport on the blood-brain barrier (BBB), and is mainly responsible for transporting Aβ from peripheral blood to the brain. Under normal physiological conditions, RAGE on the BBB can bind Aβ in the peripheral blood circulation at nanomolar levels and transport it into the brain. After blocking the outward transport of Aβ, all soluble Aβ in the brain is filled by Aβ in the peripheral circulation after 40 min of inward transport by RAGE, and the expression of RAGE on the BBB in AD is significantly up-regulated (Slowi A, et al, MOL NERURODEGENER 20127:55). The up-regulation of RAGE expression can increase Aβ into the brain and promote the deposition of Aβ in the brain. It can be seen that RAGE plays an extremely important role in Aβ transport in AD.

Aβ-RAGE signaling pathway and BBB damage: RAGE interacts with Aβ on the BBB, causing damage to microvascular endothelial cells, and positively feedbacks the expression of RAGE through NF-kB, further promoting the deposition of Aβ into the brain, causing cell damage, and destroying the blood-brain barrier. The tight junction system (BBB-TJ) (Origlia N et al, ANN N YACID SCI, 200B, 1126:147-151) mainly destroys the blood-brain barrier through the following three ways: (1) Aβ-RAGE-Ca ²⁺ -Calcineurin Signaling pathway: TJ signaling is mainly regulated by ^{Ca 2+ ,} which participates in the formation of various intercellular connections and plays an important role in maintaining the normal function of TJ. Aβ-RAGE can directly and indirectly cause Ca ²⁺ to flow into the cytoplasm, and the change of Ca ²⁺ concentration will further affect the formation of TJ, resulting in the destruction of the stability of TJ structure. In addition, calcineurin is the only serine/threonine protein phosphatase regulated by Ca ² /calmodulin. In vitro experiments found that Aβ-RAGE damages TJ protein, increases BBB permeability and can increase endothelial cell Ca ²⁺ concentration, inhibition of RAGE and calcineurin can block Aβ-induced TJ damage and improve BBB permeability (KOO SY, et al JNeurosci 2012, 32(26): 8845-8854). In summary, Ca ²⁺ is involved in the damage of the BBB, and CaNZ is an important phosphorylase of the Ca ²⁺ /CaM pathway, and its activity is regulated by Aβ-RAGE. (2) Aβ-RAGE-MMPs signaling pathway: The increase of BBB permeability is related to the increase of MMPs. After inhibiting the MMPs gene, it has a protective effect on the brain, and mainly reduces the BBB permeability (HuQ, et al Exp neurol, 2009 , 216(1) 35-46). BBB permeability increased in AD patients, ECs MMP-2, MMP-9 expression significantly increased, claudine-1, claudin-5 expression decreased, Aβ-RAGE interaction increased endothelial cell MMP-2, MMP-9 expression, decreased claudin- 1. The content of claudin-5 can damage the TJ structure, and blocking the combination of Aβ and RAGE can inhibit the expression of MMPs in brain endothelial cells induced by Aβ. It can be seen that the damage of Aβ to BBB is related to the increased expression of MMP-2 and MMP-9 induced by RAGE. (3) Aβ-RAGE-endothelial cell inflammation: The interaction between Aβ and RAGE E triggers the generation of ROS and activates the inflammatory pathway, activates NF-kB, and NF-kB acts as a positive feedback to promote the upregulation of RAGE expression, and the generation of ROS also increases It will amplify and aggravate the inflammatory process, cause damage and apoptosis of endothelial cells, and eventually destroy the integrity of the BBB, causing damage to the BBB.

Aβ can up-regulate the level of RAGE in the brain, and can activate intracellular pathways in neurons through RAGE, causing oxidative stress and inflammatory damage. Therefore, RAGE can not only promote the deposition of Aβ by transporting Aβ into the brain, but also activate the inflammatory response to damage brain tissue and promote the pathogenesis of AD. In normal healthy human brain tissue, the expression of RAGE is very small, but in AD patients, RAGE is widely distributed and its expression is significantly up-regulated. Moreover, the expression of RAGE was significantly increased in tissues with more Aβ deposition, and the expression of RAGE was lower in tissues with less Aβ deposition. Tissues with high expression of RAGE have serious inflammatory damage. The combination of Aβ and RAGE causes oxidative stress, activates the inflammatory response, causes mitochondrial damage, changes the mitochondrial membrane potential, and increases mitochondrial permeability. Mitochondria can mediate the occurrence of apoptosis pathway and release Cytochrome C leads to the caspase cascade reaction, further strengthens the PI3K/AKT pathway, induces apoptosis, and can significantly activate nuclear transcription factor-kB to form a positive feedback to promote more RAGE expression, further aggravating neuronal damage. The specific mechanism of oxidative stress caused by Aβ through mitochondria: (1) Mitochondrial dysfunction caused by mitochondrial dysfunction. In APP transgenic mice, the activity of cytochrome C was found to be weakened. Aβ is also a potent inhibitor of complex IV. AD patients Aβ affects mitochondrial function by inhibiting different enzymes in the process of energy production in mitochondria, causing mitochondrial dysfunction. (2) Lead to oxidative stress and ROS production: In normal body cells, a small amount of ROS will be produced, and a small amount of ROS has no obvious effect on cells, but in AD patients, neuron cells will produce a large amount of ROS. A small amount of ROS plays the role of signal transduction, and too much ROS will damage the body, disrupt the stability of mitochondrial membrane potential, and then produce apoptosis-inducing factors, activate cell apoptosis, and eventually cause cell death. Mitochondria are the energy-generating parts of cells. Mitochondria function abnormally, causing energy metabolism disorders, which further affect cell functions. Studies have also found that mitochondria in cells of AD patients are significantly reduced (Shacka JJ, et al, Frontbiosci, 2008 718-736). (3) Oxidative stress can lead to abnormal mitochondrial protein structure: another important way for ROS to damage mitochondria—mitochondrial protein damage. Mitochondrial protein misfolding and aggregation can lead to severe mitochondrial dysfunction and even lead to mitophagy. (4) ROS can damage mitochondrial DNA: Human mitochondrial DNA is a double-stranded, circular structure with a size of about 16.5kb, including protein synthesis genes and oxidative phosphorylation component synthesis genes. In AD patients, both nuclear DNA and mitochondrial DNA are oxidatively modified. There is an increase. In fact, 3 of the 13 subunits of the respiratory chain complex are encoded by mitochondrial DNA, so mitochondrial DNA oxidative damage can lead to damage to the structure of the mitochondrial complex, which in turn leads to abnormal mitochondrial function (Valavanidis A et al, JESHCECER 2009, 120-139).

Microglia are the most important immune cells in the brain. Under normal conditions, membrane surface receptors such as RAGE and CD47 play a major role in the endocytosis of Aβ, which is the main way for Aβ to be cleared in the brain. Nerve cells have a good protective effect, but in AD patients, due to the massive accumulation of Aβ in the brain, which exceeds the endocytic capacity of microglial cells, excessive Aβ interacts with the microglial membrane surface receptor RAGE to activate the cell Microglia release a large number of pro-inflammatory factors to damage nerve cells and disrupt the stability of the brain environment. Like neurons, microglia can also activate NF-kB through Aβ-RAGE, upregulate RAGE, and form a positive feedback.

With the acceleration of the aging society in our country, the incidence and prevalence of neurodegenerative diseases are increasing year by year. Among the elderly after the age of 65, the incidence of AD doubles every 5 years, and among the elderly over 85 20% - 50% suffer from different degrees of AD. AD has brought huge economic losses to the society, and brought immeasurable burdens to patients and their families. Therefore, it is of great social and practical significance to study specific drugs for the treatment of AD.

Contents of the invention

In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a specific antagonistic peptide for receptors of advanced glycation end products, which can specifically bind to RAGE and inhibit the action of RAGE.

Another object of the present invention is to provide derivatives of the above-mentioned specific antagonistic peptides for receptors for advanced glycation end products, which can specifically bind to RAGE and inhibit the action of RAGE.

Another object of the present invention is to provide the above-mentioned advanced glycation end product receptor-specific antagonistic peptide and the application of its derivatives.

The object of the present invention is achieved through the following technical solutions:

A specific antagonistic peptide for receptors of advanced glycation end products, the amino acid residue sequence of which is: Ala-Pro-Asp-Thr-Lys-Thr-Gln (APDTKTQ);

The derivative of the specific antagonistic peptide of the advanced glycation end product receptor is the specificity of the advanced glycation end product receptor on the amino acid side chain group of the specific antagonistic peptide of the advanced glycation end product receptor. The product obtained by routinely modifying the amino-terminal or carboxyl-terminal of the antagonistic peptide fragment, or the product obtained by attaching a tag for polypeptide or protein detection or purification to the specific antagonistic peptide of the receptor for advanced glycation end products;

The conventional modification is preferably amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification , polyethylene glycol PEG modification or immobilization modification, etc.;

The tag is His ₆ , GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or Profinity eXact, etc.;

The derivative of the specific antagonistic peptide of the advanced glycation end product receptor is preferably the second amino acid residue of the specific antagonistic peptide of the advanced glycation end product receptor is a D-configuration proline, and the terminal Carry out amidation modification, namely Ala-(d)Pro-Asp-Thr-Lys-Thr-Gln-NH ₂ ;

The preparation of the specific antagonistic peptide of the advanced glycation end product receptor and its derivatives is carried out by using known methods in the prior art. It can be chemically synthesized by an automatic polypeptide synthesizer, or can be synthesized by short peptide Sequence deduce nucleotide sequence, then clone into expression vector for biosynthesis;

The specific antagonistic peptide of the advanced glycation end product receptor and its derivatives can be applied to the preparation of drugs for the treatment of neurodegenerative diseases;

A drug for treating neurodegenerative diseases, comprising at least one of the above-mentioned receptor-specific antagonist peptides for advanced glycation end products and derivatives of receptor-specific antagonist peptides for advanced glycation end products;

The drug for treating neurodegenerative diseases may also contain one or at least two pharmaceutically acceptable carriers;

The carrier is preferably a slow-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption accelerator, adsorption carrier, surfactant or lubricant, etc.;

The medicine for treating neurodegenerative diseases can be further made into injections, tablets, granules, capsules and other forms, and medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy;

The neurodegenerative disease is preferably Alzheimer's disease (AD) or Parkinson's disease (PD);

Compared with the prior art, the present invention has the following advantages and effects:

The present invention provides a specific antagonistic peptide of advanced glycation end product receptor and its derivatives, and the antagonistic peptide can specifically combine with RAGE. The research results show that the specific antagonistic peptide of the advanced glycation end product receptor screened by the present invention can antagonize the interaction between RAGE and Aβ on the endothelial cells of the blood-brain barrier, and can be used for the prevention and treatment of neurodegenerative diseases, such as: Al Alzheimer's disease and Parkinson's disease.

Description of drawings

Fig. 1 is the mass detection spectrum of the specific antagonistic peptide of the advanced glycation end product receptor.

Fig. 2 is an analysis diagram of the results of the specific binding of the specific antagonistic peptide of the receptor for advanced glycation end products to SH-SY5Y cells. Among them, A: the specific antagonistic peptide of the advanced glycation end product receptor does not bind to 3T3 cells; B: the specific antagonistic peptide of the advanced glycation end product receptor specifically binds to SH-SY5Y cells.

Figure 3 is an analysis diagram of the results of the effect of the specific antagonistic peptide of the receptor for advanced glycation end products on cell survival, wherein, A: The specific antagonistic peptide of the receptor for advanced glycation end products has no significant effect on the survival rate of normal cells 3T3 Effect; B: The result of AD model constructed by Aβ; C: The result of the effect of the specific antagonist peptide of the receptor for advanced glycation end products on the survival rate of AD model cells.

Fig. 4 is an analysis diagram of the results of reduction of superoxide in AD model cells by the specific antagonistic peptide of the receptor for advanced glycation end products.

detailed description

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

It should be noted that those skilled in the art should understand that, unless otherwise specified, the reagents and enzymes used in the following examples are reagents or enzymes of analytical grade commercially available from reagent companies.

Example 1 Organic Synthesis of Specific Antagonist Peptide for Advanced Glycation End-Product Receptor

Adopt CS936 peptide synthesizer (USA CSBio company), Fmoc solid-phase synthesis method synthesis (synthesized by Shanghai Qiangyao biological company), the synthesis process includes the following steps:

(a) Deprotection: use piperidine (piperidine, Shanghai Ziyi Reagent Factory) solution to remove the protecting group of the amino group;

(b) Activation and cross-linking: the carboxyl group of the next amino acid is activated and dissolved by the activator HBTU (HCTU/HITU)+NMM, and the activated monomer reacts with the free amino group to form a peptide bond;

(c) cycle: (a), (b) two-step reaction cycle repeatedly until the entire peptide chain is synthesized;

(d) Elution and deprotection: According to the different residues contained in the peptide chain, it is eluted from the column with different deresin solvents, and its protective group is eluted and deprotected by a deprotecting agent (TFA). ;

(e) The synthesized short peptide was purified by Varian Prostar210 purification column (VARIAN Company, USA), and UV-Vis-detector: Varian Prostar345 (VARIAN Company, USA) was used for detection during the purification process;

(f) Adopt System Gold HPLC (Beckman Company of the United States) to verify that the purity reaches more than 99%;

(g) Thermo Finnigan LCQ deca XP plus (Thermo Company, USA) was used to detect the molecular weight of the synthetic short peptide. Accompanying drawing 1 shows the detection result of the molecular weight of the Ala-Pro-Asp-Thr-Lys-Thr-Gln short peptide, which is consistent with the theoretical value, and the specific antagonistic peptide of the advanced glycation end product receptor is obtained.

Example 2 The specific antagonistic peptide of the advanced glycation end product receptor binds specifically to RAGE on the cell membrane surface of cells with high expression of RAGE

The advanced glycation end product receptor-specific antagonist peptide prepared in Example 1 was labeled with FITC (Shanghai Qiangyao Biological Co., Ltd.). Confocal plates were seeded with SH-SY5Y-app cells (ATTC, Manassas, VA, USA), 10,000 cells per plate, and cultured in a carbon dioxide incubator (5% CO ₂ , 37°C) for 24 hours, then washed three times with PBS, poly Fix with formaldehyde for 15 min, then wash with PBS for 3 times, block with BSA for 1.5 h, then incubate with FITC-labeled advanced glycation end product receptor-specific antagonist peptide at a final concentration of 100 μmol/mL for 2 h at room temperature, wash with PBST three times, DAPI Nuclear staining was observed with a laser confocal scanning microscope (Zeiss, Germany), and 3T3 cells (Shanghai Cell Bank) were used as a negative control. The results are shown in Figure 2.

Figure 2A is the experimental results of the control cell 3T3 cells, indicating that the specific antagonist peptide of the receptor for advanced glycation end products cannot bind to the surface of 3T3 cells that do not express RAGE, and Figure 2B is the SH-SY5Y-app cells that highly express RAGE The experimental results show that the specific antagonist peptide of the advanced glycation end product receptor can specifically bind to the surface of SH-SY5Y-app cells.

Example 3 Advanced Glycation End-Product Receptor Specific Antagonist Peptide Improves the Survival Rate of AD Model Cells

(1) 3T3 cells were plated on a 96-well plate, with 10,000 cells per well, cultured for 24 hours (5% CO ₂ , 37°C), removed the original medium, and added 100 μL of different final concentrations (0, 0.1, 1, 10, 100 μM ) specific antagonistic peptide (prepared in Example 1) of the advanced glycation end product receptor, cultured for 48 hours, and the cell viability was measured by MTT method, the results are shown in Figure 3A.

(2) Construction of AD model by Aβ: SH-SY5Y-app cells were plated in a 96-well plate, with 10,000 cells per well, cultured for 24 hours (5% CO ₂ , 37°C), the original medium was removed, and 100 μL of different final concentrations ( 0, 25, 50, 100, 200, 400, 800, 1600 μM) of Aβ (Aβ was diluted with serum-free medium), cultured for 48 hours, and the cell viability was measured by MTT method, the results are shown in Figure 3B.

(3) SH-SY5Y-app cells were plated on a 96-well plate, with 10,000 cells per well, cultured for 24 hours (5% CO ₂ , 37°C), removed the original medium, and added 100 μL of different final concentrations (0, 0.1, 1 , 10, 100 μM) of the specific antagonistic peptide of the advanced glycation end product receptor (prepared in Example 1), pre-protected for 2h, and then adding 100 μL of Aβ with an initial concentration of 800nmol/mL to each well (Aβ was cultured with serum-free base dilution), cultured for 48 hours, and the cell survival rate was measured by MTT method, wherein, the control group did not add the specific antagonistic peptide of the advanced glycation end product receptor and Aβ, and replaced it with the same volume of medium; the treatment group only added Aβ was AD model group (mod group), the results are shown in Figure 3C.

Figure 3A shows that for normal 3T3 cells, the specific antagonistic peptide of the advanced glycation end product receptor does not affect the cell survival rate and has no cytotoxicity; Figure 3B shows that the cell survival rate decreases with the increase of Aβ concentration; Figure 3C shows that, For Aβ-induced nerve cell injury, when the concentration of the specific antagonist peptide of the receptor for advanced glycation end products reached above 10 μM, the cell survival rate was significantly increased.

Example 4 The specific antagonistic peptide of the receptor for advanced glycation end products reduces ROS in AD model cells

200,000 SH-SY5Y-app cells in the logarithmic phase were inoculated into 6-well plates, cultured for 24 hours until 85% of the wells were covered with cells, and late sugars with different final concentrations (0, 0.1, 1, 10, 100 μM) were added to each well The specific antagonist peptide (prepared in Example 1) of the ylated end product receptor was pre-protected for 2 h, and then Aβ (Aβ was diluted with serum-free medium) with a final concentration of 400 nmol/mL was added to each well and cultured for 48 h. DCFH-DA detected the cells Internal ROS, wherein, the control group did not add the specific antagonist peptide of the receptor for advanced glycation end products and Aβ, and replaced it with the same volume of medium; the results are shown in Figure 4.

Figure 4 shows that the specific antagonistic peptide of the receptor for advanced glycation end products can significantly reduce the reactive oxygen species produced by the oxidative stress induced by Aβ.

The above-mentioned examples illustrate that the specific antagonistic peptide of the advanced glycation end product receptor involved in the present invention can be obtained by chemical synthesis. The short peptide has no cytotoxicity to normal cells and has a good The inhibitory effect of can be applied to the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD).

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (4)

1. a kind of specific antagonist peptide of Advanced Glycation End Product Receptors is in the medicine for preparing treatment nerve degenerative diseases Application, it is characterised in that its amino acid residue sequence is：Ala-Pro-Asp-Thr-Lys-Thr-Gln.

2. the specific antagonist peptide of Advanced Glycation End Product Receptors according to claim 1 is preparing treatment neurological Application in the medicine of property disease, it is characterised in that：

The medicine of described treatment nerve degenerative diseases contains the pharmaceutically acceptable carrier of one kind either at least two.

3. the specific antagonist peptide of Advanced Glycation End Product Receptors according to claim 2 is preparing treatment neurological Application in the medicine of property disease, it is characterised in that：

Described carrier is sustained release agent, excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, absorption load Body, surfactant or lubricant.

4. the specific antagonist peptide of the Advanced Glycation End Product Receptors according to any one of claims 1 to 3 is controlled in preparation Treat the application in the medicine of nerve degenerative diseases, it is characterised in that：

Described nerve degenerative diseases are Alzheimer disease or Parkinson's.