WO2024029992A1 - Amyloid beta-specific peptide sma_04088-2 and composition for treating alzheimer's disease comprising same - Google Patents

Abstract

The present invention relates to a peptide capable of directly binding to amyloid beta protein Aβ42, which is known to be a causative agent of Alzheimer's disease, and a composition for the treatment and prevention of Alzheimer's disease, comprising the peptide. When peptide SMA_04088-2 of the present invention is used, the fibrilization and oligomerization of amyloid beta can be prevented, and thus, the peptide SMA_04088-2 can be used in the treatment and prevention of amyloid diseases such as Alzheimer's disease. In addition, the peptide SMA_04088-2 can be used to detect amyloid beta, and thus can be used in the diagnosis of amyloid diseases such as Alzheimer's disease.

Description

Amyloid beta-specific peptide SMA_04088-2 and composition for treating Alzheimer's disease containing the same

This invention was made under project number 2021R1A6A1A03038996 under the support of the Ministry of Science and ICT of the Republic of Korea. The research management agency for the project is the National Research Foundation of Korea, the research project name is "Establishment of a science and engineering academic research base," and the research project name is "Diagnosis of geriatric diseases." and development of treatment technology", the project carrying out institution is Gachon University, and the research period is 2021.06.01-2030.05.31.

This invention was made under project number 201703052 under the support of the Ministry of Oceans and Fisheries of the Republic of Korea. The research management agency for the project is the Korea Institute for the Promotion of Oceans and Fisheries Science and Technology, the research project name is "Post Genome Multi-Ministry Genome Project", and the research project name is "Marine and Fisheries Life Science." “Engineering Technology Development Project”, the project implementing agency is Korea Institute of Ocean Science and Technology, and the research period is 2017.04.01 to 2022.12.31.

This patent application claims priority to Korean Patent Application No. 10-2022-0097554 filed with the Korean Intellectual Property Office on August 4, 2022, the disclosure of which is incorporated herein by reference.

The present invention relates to a peptide capable of directly binding to amyloid beta protein Aβ42, known to be the causative agent of Alzheimer's disease, and a composition for the treatment and prevention of Alzheimer's disease containing a peptide.

The development of treatments for Alzheimer's disease by large global pharmaceutical companies is failing one after another. Until now, most treatments for Alzheimer's disease have been developed focusing on antibodies that bind to amyloid beta protein (Aβ) and tau protein (tau), but they still have many limitations.

In the case of amyloid beta protein (Aβ), oligomers are created by oligomerizing with each other, and when aggregation continues, fibrilization progresses to form a fibril form. There is ongoing debate as to which form of amyloid beta protein, oligomer or fibril form, causes toxicity to nerve cells. Considering the research results to date, the prevailing argument is that oligomers can cause toxicity to cells.

A representative method for screening compound-based drug candidates is to confirm the effect of inhibiting fibrilization of amyloid beta protein. However, since fibrosis of amyloid beta protein is also considered a mechanism to protect against cytotoxicity, it may not be an appropriate screening method for drug candidates. Additionally, there is a problem that the fibrosis-inhibiting effect of amyloid beta protein may occur by increasing the stability of oligomers with toxic properties.

As amyloid beta oligomer is known to be a major toxic substance in Alzheimer's disease, the importance of diagnosis or treatment targeting it is highlighted. High efficiency requires high binding affinity to amyloid beta oligomers, and for treatment, substances that are non-toxic and easily decomposed must be screened. From this perspective, naturally derived peptides can compensate for the shortcomings of toxicity of synthetic compounds because they are easily decomposed in vivo, and because they can be synthesized directly, they have the advantage of being low-cost, unlike antibodies.

The present inventors made extensive efforts to search for a peptide that directly binds to amyloid beta protein and prevents fibrosis and oligomerization of amyloid beta protein. As a result, it was discovered that peptide SMA_04088-2 has a therapeutic effect on amyloid disease by preventing fibrosis and oligomerization of amyloid beta protein, thereby completing the present invention.

Therefore, an object of the present invention is to provide a polypeptide comprising the amino acid sequence of peptide SMA_04088-2.

Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of peptide SMA_04088-2.

Another object of the present invention is to provide a recombinant vector containing a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

Another object of the present invention is to provide a host cell transformed with a recombinant vector containing a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating amyloid beta-related neurodegenerative disease, including a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

Another object of the present invention is to provide a composition for detecting amyloid beta protein containing a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

Another object of the present invention is amyloid beta, comprising the step of detecting a peptide-amyloid beta protein complex formed by contacting a sample sample with a composition for detecting amyloid beta protein containing a polypeptide containing the amino acid sequence of peptide SMA_04088-2. It provides a method for detecting proteins.

The present invention provides inventions 1 to 12 below.

1. Polypeptide containing the amino acid sequence of SEQ ID NO: 1.

2. A nucleic acid molecule containing a nucleotide sequence encoding a polypeptide of 1.

3. The nucleic acid molecule according to 2, wherein the nucleotide sequence is the nucleotide sequence of SEQ ID NO: 2.

4. Recombinant vector containing 2 nucleic acid molecules.

5. Host cells transformed with the recombinant vector of 4.

6. A pharmaceutical composition for preventing or treating amyloid beta-related neurodegenerative disease, comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 1.

7. The composition of claim 6, wherein the polypeptide inhibits fibrosis and oligomerization of amyloid beta.

8. The composition according to clause 6, wherein the neurodegenerative disease is any one disease selected from the group consisting of Alzheimer's disease, amyloid angiopathy, amyloid stroke, systemic amyloid disease, and Parkinson's disease.

6. A composition for detecting amyloid beta protein containing a polypeptide containing the amino acid sequence of SEQ ID NO: 1.

7. A method for detecting amyloid beta protein comprising the step of detecting a peptide-amyloid beta protein complex formed by contacting the composition of 6 with a sample sample.

8. A method for preventing or treating amyloid beta-related neurodegenerative disease, comprising administering to a subject the polypeptide of 1 or the composition of any one of 6 to 8.

9. Use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 to prevent or treat amyloid beta-related neurodegenerative disease.

10. Use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 in the manufacture of a drug for preventing or treating amyloid beta-related neurodegenerative disease.

11. Kit for detecting amyloid beta protein comprising the composition of 6.

12. A method for diagnosing amyloid beta-related neurodegenerative disease, comprising the step of detecting a peptide-amyloid beta protein complex formed by contacting the polypeptide of 1 or the composition of 6 with a sample sample.

According to one aspect of the present invention, the present invention provides a polypeptide comprising the amino acid sequence of peptide SMA_04088-2.

In one embodiment of the present invention, the peptide SMA_04088-2 includes a polypeptide containing the amino acid sequence of SEQ ID NO: 1.

As used herein, a polypeptide may include at least one additional amino acid at the C-terminus and/or N-terminus of the polypeptide. The additional amino acid residues may be added individually or collectively for the purpose of, for example, improving productivity, purification, stabilization in vivo or in vitro, coupling of the complex, or detection. For example, the polypeptide may additionally include a cysteine residue at the C-terminus and/or N-terminus of the polypeptide. Additional amino acid residues may provide a “tag” for purification or detection of the polypeptide, for example for interaction with a specific antibody. For the His6 tag, for immobilized metal affinity chromatography (IMAC), tags such as His6 tag, (HisGlu)3 tag ("HEHEHE" tag) or "myc" (c-myc) tag or "FLAG" tag are used. can be provided.

The polypeptide containing the amino acid sequence of SEQ ID NO: 1 of the present invention is interpreted to include the amino acid sequence of SEQ ID NO: 1, and also includes a sequence showing substantial identity with the sequence of SEQ ID NO: 1. The above substantial identity is preferably achieved by aligning the sequence of the present invention and any other sequence to correspond as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art. It means a sequence showing 80% homology, more preferably at least 85% homology, even more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are discussed in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from NBCI (National Center for Biological Information), etc., and can be accessed on the Internet using blastp, blasm, It can be used in conjunction with sequence analysis programs such as blastx, tblastn, and tblastx.

As the polypeptide used in the present invention, a biological functional equivalent that is an amino acid sequence variant that exhibits the same biological activity as the polypeptide containing the amino acid sequence of SEQ ID NO: 1 of the present invention can also be used. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

In introducing mutations, the hydrophobic index of the amino acid (hydropathic idex) may be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is very important in imparting interactive biological functions to proteins. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobic index. When introducing a mutation with reference to the hydrophobicity index, substitution is made between amino acids showing a difference in hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Meanwhile, it is also well known that substitution between amino acids with similar hydrophilicity values results in proteins with equal biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0± 1); glutamate (+3.0± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing a mutation with reference to the hydrophilicity value, substitution is made between amino acids that show a difference in hydrophilicity value, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

According to another aspect of the invention, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of peptide SMA_04088-2.

In one embodiment of the present invention, the nucleotide sequence is the nucleotide sequence of SEQ ID NO: 2.

As used herein, the term "nucleic acid molecule" is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also those with modified sugars or base sites. Also includes analogues (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

It is clear to those skilled in the art that the nucleotide sequence encoding the polypeptide of the present invention is sufficient to be the nucleotide sequence encoding the amino acid sequence of the peptide SMA_04088-2, and is not limited to any specific nucleotide sequence.

This is because even if a mutation in the nucleotide sequence occurs, there are cases in which no change occurs in the protein sequence when the mutated nucleotide sequence is expressed as a protein. This is called codon degeneracy. Accordingly, the nucleotide sequence can be either a functionally equivalent codon, or a codon encoding the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine or serine), or a codon encoding a biologically equivalent amino acid. It contains a nucleotide sequence containing.

According to a specific embodiment of the present invention, the sequence of the polypeptide containing the amino acid sequence of the peptide SMA_04088-2 of the present invention is listed in the sequence list attached to this specification.

A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of peptide SMA_04088-2 is interpreted to also include a nucleotide sequence showing substantial identity to the above-described nucleotide sequence. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. refers to a nucleotide sequence that exhibits homology, more preferably at least 90% homology, and most preferably at least 95%, 97%, 98%, or 99% homology.

Considering the mutations having the above-mentioned biological equivalent activity, the nucleic acid molecule containing the nucleotide sequence encoding the polypeptide containing the amino acid sequence of the peptide SMA_04088-2 of the present invention is substantially identical to the sequence listed in the sequence listing ( It is interpreted to include sequences showing substantial identity. The above substantial identity is at least 61% when aligning the sequence of the present invention and any other sequence to correspond as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. It means a sequence showing homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are discussed in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc., and can be accessed on the Internet using blastp, blastn, It can be used in conjunction with sequence analysis programs such as blastx, tblastn, and tblastx. BLAST can be accessed through the BLAST page on the ncbi website. The sequence homology comparison method using this program can be found on the BLAST help page of the ncbi website.

According to another aspect of the present invention, the present invention provides a recombinant vector containing a nucleic acid molecule encoding a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

As used herein, the term “vector” refers to a means for expressing a gene of interest in a host cell, including a plasmid vector; cosmid vector; and viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors.

According to a specific embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of the peptide SMA_04088-2 is operatively linked to the promoter of the vector. ).

As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, thereby regulating said nucleic acid sequence. The sequence will regulate transcription and/or translation of the other nucleic acid sequences.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods are disclosed in Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) , this document is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for gene cloning or a vector for protein expression. Additionally, the vector of the present invention can be constructed using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and eukaryotic cells are used as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, beta actin promoter, human haroglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, HIV The LTR promoter of Moloney virus, the promoter of Epstein-Barr virus (EBV), and the promoter of Roussoma virus (RSV) can be used, which generally terminate transcription.

It has a polyadenylation sequence as a sequence.

The vector of the present invention may be fused with other sequences to facilitate purification of the polypeptide or protein expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6) (His(hexahistidine; Quiagen, USA).

Meanwhile, the expression vector of the present invention contains antibiotic resistance genes commonly used in the art as selection markers, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, and neomycin. and a resistance gene to tetracycline.

According to another aspect of the present invention, the present invention provides a host cell transformed with the above recombinant vector.

Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell can be used. For example, suitable eukaryotic host cells for the vector include monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, \138, and baby hamster kidney (BHK). : baby hamster kidney) cells, MDCK, myeloma cell line, HuT 78 cells, and HEK-293 cells.

As used herein, the terms “transformed,” “transduced,” or “transfected” refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A “transformed”, “transduced” or “transfected” cell is a cell that has been transformed, transduced or transfected with an exogenous nucleic acid, including that cell and progeny cells resulting from subculture thereof.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating amyloid beta-related neurodegenerative disease, comprising a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

In one embodiment of the present invention, the polypeptide inhibits fibrosis and oligomerization of amyloid beta.

The term 'amyloid beta' herein refers to amyloid precursor protein (APP) consisting of 39 to 43 amino acids produced by sequential hydrolysis by beta and gamma secretase (β-, γ-secretase). Refers to multiple proteins.

The term 'amyloid beta 42 (Aβ42)' in this specification refers to an amyloid beta protein with an Ala C-terminus that is highly cohesive and is known to accumulate predominantly in the brains of Alzheimer's disease patients from early on.

As used herein, the term amyloid beta 'oligomer' refers to a soluble or insoluble form in which amyloid beta monomers are aggregated together.

The term amyloid beta 'oligomerization' as used herein refers to the process of forming soluble or insoluble oligomers in which amyloid beta monomers are integrated together.

The term 'degenerative neurological disease' herein refers to a group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, amyloid angiopathy, amyloid stroke, and systemic amyloid disease. It means that it is selected from.

The term 'Alzheimer's disease (AD)' in this specification refers to the formation of amyloid plaques due to the deposition of amyloid beta in brain areas such as the cerebral cortex and hippocampus, and the protein tau binds to microtubules, resulting in neurofibrillary tangles. It refers to a degenerative neurological disease in which a neurofibrillary tangle is formed, accompanied by gradual loss of memory, decline in cognitive ability, and dementia. Clinical symptoms of Alzheimer's disease include cognitive impairment such as memory loss, language ability, decreased ability to grasp space and time, decreased judgment and ability to perform daily activities, and gait and mobility problems.

The term 'prophylaxis' herein refers to the prevention or protective treatment of a disease or disease state. The term 'treatment' herein means reducing, suppressing, soothing or eradicating a disease state.

The pharmaceutical composition of the present invention includes powders, granules, tablets, coated tablets, pills, sugar-coated tablets, capsules, solutions, suspensions, gels, syrups, slurries, suppositories, enemas, emulsions, pastes, ointments, It may be formulated as a cream, lotion, powder, spray, or suspension.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. For example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, mannitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl. These include pyrlidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable harm/risk ratio applicable to medical treatment, and the effective dose level refers to the type of patient's disease, severity, activity of the drug, and It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The amount of the composition used may vary depending on the patient's age, gender, and weight, but a sufficient amount can be administered once or several times a day to ensure that the peptide reaches a blood concentration useful for the treatment of amyloid disease.

The dosage of the composition may be increased or decreased depending on the route of administration, degree of disease, gender, weight, age, etc. Accordingly, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including intracerebral administration, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, and intrarectally. It can be administered by insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of Alzheimer's dementia, and “treatment” refers to any action that improves or beneficially changes Alzheimer's dementia by administering the pharmaceutical composition according to the present invention. , “Improvement” means any action that reduces parameters related to Alzheimer's dementia, such as the severity of symptoms, by administering the composition according to the present invention.

According to another aspect of the present invention, the present invention provides a composition for detecting amyloid beta protein comprising a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

The composition for detecting amyloid beta protein of the present invention may further include additives that can be included in conventional compositions for detecting residual drugs, such as auxiliary substances for maintaining the activity of the peptide and confirming the binding between the peptide and amyloid beta protein.

The composition for detecting amyloid beta protein of the present invention includes tools for collecting samples of test subjects or applying diagnostic reagents, reagents or devices for confirming the binding of peptide SMA_04088-2 and amyloid beta protein, etc. from biological samples using peptides. Tools, devices, or reagents that can be included in a typical diagnostic composition for confirming the presence or absence of amyloid beta protein may be further included.

Tools/reagents that may be included in the detection composition include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, solubilizers, detergents, buffers, stabilizers, etc. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, It may be polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, polymers such as magnetic particles made by plating metal on latex, other paper, glass, metal, agarose, and combinations thereof.

As used herein, the term 'multimer detection system (MDS)' refers to a method for differentially detecting the multimer form from the monomer form of a multimer-forming polypeptide. More specifically, it may be a detection method disclosed in Republic of Korea Patent No. 10-0987639.

The term 'Thioflavin T (ThT) assay' herein refers to an analysis technique used to quantify the degree of fibrosis formation and inhibition of amyloid protein in the presence of anti-amyloidogenic compounds.

According to another aspect of the present invention, the present invention provides a method for detecting amyloid beta protein, comprising the step of detecting a peptide-amyloid beta protein complex formed by contacting the composition for detecting amyloid beta protein with a sample sample.

According to one embodiment of the present invention, the detecting step can be performed through any experimental method that can confirm the binding signal of protein and peptide, such as ELISA or surface plasma resonance (SRP) technique.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

(b) The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of peptide SMA_04088-2.

(c) The present invention provides a recombinant vector containing a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

(d) The present invention provides a host cell transformed with a recombinant vector containing a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

(e) The present invention provides a pharmaceutical composition for preventing or treating amyloid beta-related neurodegenerative disease, comprising a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

(f) The present invention provides a composition for detecting amyloid beta protein containing a polypeptide containing the amino acid sequence of peptide SMA_04088-2.

(g) The present invention relates to amyloid beta protein, comprising the step of detecting a peptide-amyloid beta protein complex formed by contacting a sample sample with a composition for detecting amyloid beta protein containing a polypeptide containing the amino acid sequence of peptide SMA_04088-2. Provides a detection method.

(h) When using the peptide SMA_04088-2, which directly binds to the amyloid beta protein of the present invention, fibrosis and oligomerization of amyloid beta can be prevented, so it can be used for the treatment and prevention of amyloid diseases such as Alzheimer's. Additionally, since it can be used to detect amyloid beta, it can be used to diagnose amyloid diseases such as Alzheimer's.

Figure 1 shows the structure and amino acid sequence of peptide SMA_04088-2. Peptide SMA_04088-2 of Figure 1 contains six cysteines and a total of three disulfide bonds. The disulfide bond is formed between the first Cys and the sixth Cys, the second Cys and the fourth Cys, and the third Cys and the fifth Cys, respectively. Specifically, in the peptide of FIG. 1, Cys 1 and Cys 39, Cys 10 and Cys 32, and Cys 19 and Cys 36 may form disulfide bonds, respectively.

Figure 2 shows the fibrosis inhibition effect of amyloid beta protein (Aβ) measured using Thioflovin T (ThT) assay. In the case of the peptide SMA_04088-2 treatment group, compared to the negative control in which no substance was added and the positive control in which doxorubicin, a compound previously known to inhibit amyloid beta binding, was added, amyloid It indicates that it inhibits fibrosis of beta protein.

Figure 3 shows the effect of the drug on inhibiting oligomerization of amyloid beta protein (Aβ) using a multimer detention system (MDS) assay. It indicates that peptide SMA_04088-2 inhibits oligomerization of amyloid beta protein.

Figure 4 shows direct binding between peptide SMA_04088-2 and amyloid beta 42 (Aβ42). After the peptide was hydrophilically bound to the plate surface, it was treated with Aβ42 to induce binding, and the binding was measured by ELISA using an anti-Aβ42 antibody. In both regular ELISA and malemide ELISA, peptide SMA_04088-2 shows high signal binding to Aβ42.

Figure 5 measures whether the cytotoxicity of Aβ is inhibited when treated with peptide SMA_04088-2. When neuroblastoma SH-SY5Y cells were treated with peptide SMA_04088-2 and cultured with Aβ1-42 (5M), cell survival rate increased.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1: Experimental method

1-1. Synthesis of peptide SMA_04088-2

Peptide SMA_04088-2 shown in Figure 1 was produced by requesting Shanghai royobiotech Co., Ltd. The peptide SMA_04088-2 used in the present invention is preferably synthesized by a common peptide chemical synthesis method in the art (W H Freeman and Co, Proteins; structures and molecular principles, 1983), specifically, a liquid peptide synthesis method (Solution). Phase peptide synthesis, solid-phase peptide syntheses, fragment condensation, and F-moc or T-BOC chemical methods are more preferable, and more specifically, solid-phase peptide synthesis is most preferable. do.

1-2. ThT assay

Amyloid beta was prepared by dissolving human Aβ42 (AggreSure ^™ , AnaSpec) in PBS. The peptide was added to amyloid beta, mixed with thioflavin T solution (ThT, 50 μM, Sigma Aldrich), and reacted at 37°C for 24 hours. Afterwards, the fluorescence signal was measured using a microplate reader (PerkinElmer Victor-3 ^® ). As a negative control, a mixture of thioflavin T solution and amyloid beta reacted without addition of peptides or compounds was used.

1-3. MDS assay

First, a coating buffer was prepared by diluting amyloid beta capturing antibody 6E10 (BioLegend, USA) in Sodium Carbonate-Bicarbonate at pH 9.4. The coating buffer was dispensed into a 96 well plate and incubated overnight in a 4°C refrigerator to coat the plate with the capture antibody.

Blocking buffer was dispensed onto the coated plate and incubated at room temperature for 2 hours to block. The blocking buffer is PBST containing 2% bovine serum albumin (BSA). Each sample that went through the blocking process was dispensed into a well. Afterwards, the reaction was incubated at room temperature for 1 hour.

Then, detection buffer was dispensed into each well and incubated at room temperature for 30 minutes. The detection buffer is PBST containing a detecting antibody WO2-HRP (WO2-horseradish peroxidase antibody, PeopleBio Inc., South Korea) at a concentration of 0.1 μg/ml.

TMB (3,3',5,5'-tetramethylbenzidine) solution was dispensed into each well and incubated at temperature for 30 minutes. Stop solution (H ₂ SO ₄ , 1 M) was dispensed into each well. The signal was then measured using a microplate reader (PerkinElmer Victor-3 ^® ) at a wavelength of 450 nm. A negative control also measured the signal at a wavelength of 450 nm.

1-4. ELISA

In the case of general ELISA, the polypeptide was diluted in sodium carbonate-bicarbonate at pH 9.4, dispensed onto a microplate, and then reacted overnight at 4°C. Blocking buffer was dispensed onto the coated plate and incubated at room temperature for 2 hours to block. The blocking buffer is PBST containing 2% bovine serum albumin (BSA). After the blocking process was completed, Aβ42 was treated and reacted with the polypeptide. Then, detection buffer was dispensed into each well and incubated at room temperature for 30 minutes. The detection buffer is PBST containing a detecting antibody WO2-HRP (WO2-horseradish peroxidase antibody, PeopleBio Inc., South Korea) at a concentration of 0.1 μg/ml. TMB (3,3',5,5'-tetramethylbenzidine) solution was dispensed into each well and incubated at temperature for 30 minutes. Stop solution (H ₂ SO ₄ , 1 M) was dispensed into each well. The signal was then measured using a microplate reader (PerkinElmer Victor-3 ^® ) at a wavelength of 450 nm. A negative control also measured the signal at a wavelength of 450 nm.

In the case of Maleimide ELISA, polypeptides diluted in PBS were treated on Maleimide Activated Plates (Thermo), and then reacted overnight at 4°C. Blocking buffer was dispensed onto the coated plate and incubated at room temperature for 2 hours to block. The blocking buffer is PBST containing 2% bovine serum albumin (BSA). After the blocking process was completed, Aβ42 was treated and reacted with the polypeptide. Then, detection buffer was dispensed into each well and incubated at room temperature for 30 minutes. The detection buffer is PBST containing a detecting antibody WO2-HRP (WO2-horseradish peroxidase antibody, PeopleBio Inc., South Korea) at a concentration of 0.1 μg/ml. TMB (3,3',5,5'-tetramethylbenzidine) solution was dispensed into each well and incubated at temperature for 30 minutes. Stop solution (H2SO4, 1 M) was dispensed into each well. The signal was then measured using a microplate reader (PerkinElmer Victor-3®) at a wavelength of 450 nm. A negative control also measured the signal at a wavelength of 450 nm.

1-5. ATP luminescence assay

Cells were distributed in DMEM culture medium to a concentration of 2 Х10 cells/well in a 96 well plate and stabilized overnight. Only amyloid beta was treated as a control group, and polypeptide and amyloid beta were treated as experimental groups. were treated together and reacted for 24 hours. After removing the culture medium, 100 μl of 0.5 mg/ml MTT solution was added to each well and incubated in a 37°C incubator for at least 1 hour. Then, MTT was removed and DMSO was dispensed at 200 μl each. The absorbance of formazin produced in the well was measured. Absorbance was measured at 540 nm with an ELISA reader.

Example 2: Selection and verification of candidate substances for treating Alzheimer's disease

Candidate substances that prevent oligomerization and fibrilization of amyloid beta protein, the causative agent of Alzheimer's disease, were explored. Previously, substances that prevent fibrosis of amyloid beta protein were generally selected as candidates for the treatment of Alzheimer's disease. However, considering that the oligomeric form of amyloid beta protein is pointed out to be cytotoxic and that inhibition of fibrosis of amyloid beta can occur by increasing the stability of oligomers, it is possible that it may not be possible to properly screen effective substances based on the fibrosis inhibition effect alone. There is. Therefore, in the present invention, SMA_04088-2, a candidate for the treatment of Alzheimer's disease, was selected by verifying whether it prevents both oligomerization and fibrosis of amyloid beta protein.

2-1. Verification of Aβ fibrosis inhibition effect of SMA_04088-2 through ThT assay

SMA_04088-2 was reacted with thioflavin T and Aβ through ThT assay (Thioflavin T asaay), and fluorescence was observed to confirm the fibrosis inhibition effect of Aβ according to peptide treatment. Fluorescence was measured at 5-minute intervals for 2 hours.

The results are shown in Figure 2.

As shown in Figure 2, compared to the negative control group, Aβ fibrosis was inhibited by about 55.2% in the SMA_04088-2 treatment group, confirming that the Aβ fibrosis inhibition effect of SMA_04088-2 was very excellent.

2-2. Verification of the effect of SMA_04088-2 to inhibit Aβ oligomerization through MDS assay

The inhibitory effect of Aβ oligomerization following SMA_04088-2 treatment was verified through MDS assay (Multimer Detection System assay). Samples were prepared by reacting Aβ with SMA_04088-2 and stored at -80°C. The level of Aβ oligomerization of each sample was measured through MDS-HTS.

The results are shown in Figure 3.

As shown in Figure 3, compared to the negative control group, Aβ oligomerization in the SMA_04088-2 treatment group was inhibited by about 70%, confirming that the Aβ oligomerization inhibition effect of SMA_04088-2 was very excellent.

Example 3: Direct binding measurement of Aβ and peptide SMA_04088-2 through ELISA

The absorbance was measured by treating the plate with SMA_04088-2 and Aβ through ELISA. General ELISA was performed by hydrophilic binding the peptide to the plate surface and then treating it with Aβ42 to induce binding. The binding and extent were measured using anti-Aβ42 antibody. Maleimide ELISA has a maleimide residue on the plate surface that binds to the cysteine residue of the peptide. The peptide was bound to the plate, treated with Aβ42 in the same manner to induce binding, and the presence and extent of binding was measured using an antibody.

The results are shown in Figure 4.

As shown in Figure 4, SMA_04088-2 peptide showed high Aβ42 binding ability in general ELISA and maleimide ELISA.

Example 4: Verification of cytotoxicity inhibition effect by Aβ

5,000 neuroblastoma SH-SY5Y cells were seeded in each well of a 96-well plate, treated with drugs containing Aβ and peptide SMA_04088-2, and cell survival was measured by absorbance. The untreated control group was not treated with drugs or Aβ, and the Aβ-treated control group was treated with only Aβ without drugs. The experimental group was pretreated with a compound containing 15uM of peptide SMA_04088-2 for 5 hours and then treated with Aβ1-42 (5M) and cultured.

The results are shown in Figure 5.

As shown in Figure 5, the cell survival rate in the Aβ-treated group was only 64%, but when treated together with peptide SMA_04088-2, the survival rate increased to 82%, indicating that SMA_04088-2 can inhibit cytotoxicity caused by Aβ. was able to confirm.

Claims (10)

1. A polypeptide containing the amino acid sequence of SEQ ID NO: 1.
2. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1.
3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence is the nucleotide sequence of SEQ ID NO: 2.
4. A recombinant vector containing the nucleic acid molecule of claim 2.
5. A host cell transformed with the recombinant vector of claim 4.
6. A pharmaceutical composition for preventing or treating amyloid beta-related neurodegenerative disease, comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 1.
7. The composition of claim 6, wherein the polypeptide inhibits fibrosis and oligomerization of amyloid beta.
8. The composition of claim 6, wherein the neurodegenerative disease is any one disease selected from the group consisting of Alzheimer's disease, amyloid angiopathy, amyloid stroke, systemic amyloid disease, and Parkinson's disease.
9. A composition for detecting amyloid beta protein comprising a polypeptide containing the amino acid sequence of SEQ ID NO: 1.
10. A method for detecting amyloid beta protein comprising the step of detecting a peptide-amyloid beta protein complex formed by contacting the composition of claim 9 with a sample.

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