KR102328709B1 - peptide for detecting small molecular weight amyloid beta and use thereof - Google Patents

Abstract

The present invention relates to a peptide probe for detecting amyloid beta, and more particularly, to a peptide that specifically binds to low molecular weight amyloid beta for early diagnosis of Alzheimer's disease, and to a use thereof.
In order to overcome the disadvantage that most of the diagnostic methods for Alzheimer's disease cannot be applied to early diagnosis of Alzheimer's disease, the present inventors have found that a peptide having a specific sequence obtained by screening a probe of amyloid beta selected from a random phage library is a small molecule. As it was first discovered that it can bind with high specificity to amyloid beta 42 of Because of this high level, it is expected that it will be possible to effectively diagnose Alzheimer's disease with only a small amount of diagnostic agent.

Description

Peptide for detecting small molecular weight amyloid beta and use thereof

The present invention relates to a peptide for the detection of amyloid beta, and more particularly, a peptide that specifically binds to low molecular weight amyloid beta for early diagnosis of Alzheimer's disease, and an early stage of Alzheimer's disease using the peptide for diagnostic use.

Alzheimer's disease (Alzheimer's Disease) is the most common form of neurodegenerative disease, in which the abnormal protein, amyloid beta, is coagulated amyloid plaques or senile plaques and microtubules in cells are stabilized. It has characteristics such as the generation of neurofibrillary tangles (NFTs), which are aggregates of the protein tau protein, and symptoms such as memory loss and lack of recognition. As the aging process accelerates, the number of patients with Alzheimer's disease is rapidly increasing and it is emerging as a serious social problem. However, early detection can slow the progression of Alzheimer's and improve symptoms.

Amyloid beta refers to 36 to 43 amino acid peptides that are critically involved in Alzheimer's disease as the main component of amyloid plaques found in the brain of Alzheimer's patients. The peptide is derived from amyloid precursor protein (APP), which is degraded by beta secretase and gamma secretase to produce amyloid beta. Amyloid beta molecules can aggregate to form soluble oligomers that can exist in various forms, and are produced through normal intracellular metabolism and flow into the cerebrospinal fluid in the form of water-soluble proteins. However, these functions do not work properly, so they are deposited in brain cells, which eventually interferes with nerve transmission, leading to Alzheimer's disease.

On the other hand, there are currently various methods for diagnosing Alzheimer's disease, such as a physical examination, a neurological examination, a mental state examination, a daily living function level test, a blood test, and a brain imaging test. Despite the various diagnostic methods described above, since the diagnostic method is based on the symptoms observed when the patient's disease has already progressed to a certain extent, the diagnostic method has a disadvantage in that it cannot detect Alzheimer's disease early.

Accordingly, there is an urgent need to develop a new diagnostic agent for early diagnosis of Alzheimer's disease, and there are studies related to biomarkers for the diagnosis of Alzheimer's disease (Korea Patent Publication No. 10-1240207), but peptides for early diagnosis of the disease Research on diagnostic agents, such as probes, is scarce.

In order to overcome the limitation that most of the diagnostic methods for Alzheimer's disease cannot be applied to early diagnosis of Alzheimer's disease, the present inventors have found that a peptide having a specific sequence obtained by screening a probe of amyloid beta selected from a random phage library is a small molecule. It was first identified that it can bind to amyloid beta 42 with high specificity, and the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a peptide for targeting a small molecule amyloid beta 42, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a nucleic acid molecule encoding the amino acid sequence of the peptide.

Another object of the present invention is to provide a probe for imaging a small molecule amyloid beta protein, including the peptide.

Another object of the present invention is to provide a composition for detecting low molecular weight amyloid beta protein, including the probe.

Another object of the present invention is to provide a composition for early diagnosis of Alzheimer's disease, comprising the probe.

Another object of the present invention is to provide a method for detecting a low-molecular-weight amyloid beta protein comprising the following steps.

(a) contacting the peptide according to claim 1 with a sample derived from a detection target; and

(b) determining whether the peptide is bound to amyloid beta 42, which may be included in the sample derived from the detection target.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention

As a small molecule amyloid beta 42 targeting peptide, the peptide is characterized in that it consists of the amino acid sequence of SEQ ID NO: 1, it provides a peptide.

In one embodiment of the present invention, the low molecular weight amyloid beta 42 may be a monomer or a small oligomer.

The present invention also provides a nucleic acid molecule encoding the amino acid sequence of the peptide.

The present invention also provides a probe for imaging a small molecule amyloid beta protein, including the peptide.

In one embodiment of the present invention, the peptide may be labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.

The present invention also provides a composition for detecting low molecular weight amyloid beta protein, including the probe.

The present invention also provides a composition for early diagnosis of Alzheimer's disease, comprising the probe.

The present invention also provides a method for detecting a low molecular weight amyloid beta protein, comprising the following steps.

(a) contacting the peptide according to claim 1 with a sample derived from a detection target; and

(b) determining whether the peptide is bound to amyloid beta 42, which may be included in the sample derived from the detection target.

In one embodiment of the present invention, the peptide may be labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.

In order to overcome the disadvantage that most of the diagnostic methods for Alzheimer's disease cannot be applied to early diagnosis of Alzheimer's disease, the present inventors have found that a peptide having a specific sequence obtained by screening a probe of amyloid beta selected from a random phage library is a small molecule. As it was first discovered that it can bind with high specificity to amyloid beta 42 of Because of this high level, it is expected that it will be possible to effectively diagnose Alzheimer's disease with only a small amount of diagnostic agent.

1 shows the results of taking TEM images for each time by reacting with aggregation conditions after pretreatment of amyloid beta 42. FIG.
Figure 2 shows a schematic diagram of the M13 phage display screening process for the selection of peptides that bind to amyloid beta 42.
Figure 3 shows the results of analyzing the binding force between the selected and synthesized peptide and amyloid beta 42.
4 shows the results of confirming the detection range of amyloid beta 42 through quantification of amyloid beta 42 using the selected peptide.
5 shows the results of confirming whether or not the Alzheimer's disease patient group and the non-patient group can be significantly classified when Alzheimer's disease is diagnosed using antibody-based ELISA (n: (+) = 6, (-) = 7).
6 shows the results of confirming whether it is possible to significantly classify Alzheimer's disease patient group and non-patient group when Alzheimer's disease is diagnosed using the peptide-based measurement system according to the present invention (n: (+) = 6, (-) )= 7).

Hereinafter, the present invention will be described in detail.

In order to overcome the limitation that most of the diagnostic methods for Alzheimer's disease cannot be applied to early diagnosis of Alzheimer's disease, the present inventors have found that a peptide having a specific sequence obtained by screening a probe of amyloid beta selected from a random phage library is a small molecule. It was first identified that it can bind to amyloid beta 42 with high specificity, and the present invention was completed based on this.

Accordingly, the present invention provides a peptide for targeting a low molecular weight amyloid beta protein, wherein the low molecular weight amyloid beta protein is a monomer or a small oligomer, and the peptide is a peptide comprising the amino acid sequence of SEQ ID NO: 1 , It provides a nucleic acid molecule encoding the amino acid of the peptide, and a probe for imaging a small molecule amyloid beta protein comprising the peptide.

In the present invention, "monomer" refers to a 'unit molecule' forming a polymer and is called a monomer or a unit.

In the present invention, "oligomer (oligomer)", also called an oligomer, refers to a molecular weight of about 1,000 or less. Among polymers, such as oligosaccharides, oligopeptides, and oligonucleotides, the number of monomers, which are basic structural units, is small, and the number of monomers is often 2 to 20.

The peptide according to the present invention may include a sequence showing substantial identity to the sequence set forth in SEQ ID NO: 1. The substantial identity is at least 80% when the sequence of the present invention and any other sequences are aligned to the maximum correspondence, and the aligned sequence is analyzed using an algorithm commonly used in the art. A sequence exhibiting homology, more preferably 90%, 91%, 92%, 93%, 94%, 95% homology, most preferably 96%, 97%, 98%, 99% homology. it means.

Through the examples of the present invention, it was clarified that the peptide according to the present invention can be applied to early diagnosis of Alzheimer's disease.

In one embodiment of the present invention, in order to screen a phage peptide that binds to amyloid beta 42, an M13 phage library is put into a G protein coated plate so that IgG can be directionally immobilized and amyloid beta 42 After binding to, phages expressing peptides that bind with high affinity were selected through various binding times and washing conditions. ) was obtained (see Example 2).

In another embodiment of the present invention, an anti-amyloid beta 42 IgG is immobilized on a plate with amyloid beta 42 treated by serial dilution, and the peptide according to the present invention is labeled with biotin at 450 nm. As a result of measuring the absorbance for the amount of amyloid beta 42, it was confirmed that the peptide according to the present invention can detect amyloid beta 42 at a level of ng/ml (see Example 4).

In another embodiment of the present invention, a cerebrospinal fluid (CSF) sample collected from a patient is treated on a plate immobilized with anti-amyloid beta 42 (Anti-amyloid beta 42) IgG, and biotin is added to the peptide according to the present invention. (Biotin) was used to measure the absorbance signal for each cerebrospinal fluid (CSF) sample, and compared with the Alzheimer's disease diagnosis result through PET image, the peptide according to the present invention was compared with the Alzheimer's disease patient group. It was confirmed that the patient group could be classified significantly (Example 5, Article).

The above results confirm that the peptide according to the present invention can be used for the detection of low-molecular amyloid beta 42, and it is possible to accurately distinguish the Alzheimer's disease patient group and the non-patient group compared to the conventional Alzheimer's disease diagnosis method using ELISA, which is the present invention suggests that the peptide according to the present invention can be usefully used for early diagnosis of Alzheimer's disease.

Accordingly, the present invention provides a composition for early diagnosis of Alzheimer's disease, comprising the above probe.

In another aspect of the present invention, the present invention provides a method for detecting a low molecular weight amyloid beta protein, comprising the following steps. (a) contacting the peptide according to claim 1 with a sample derived from a detection target; and (b) determining whether the peptide is bound to amyloid beta, which may be included in the detection target-derived sample.

In the present invention, the biological sample derived from the detection target in step (a) may include tissue, cells, whole blood, blood or cerebrospinal fluid, and preferably cerebrospinal fluid (CSF). It is not limited.

In the present invention, in order to easily confirm, detect and quantify whether the peptide according to the present invention binds to amyloid beta 42, the peptide according to the present invention may be provided in a labeled state. That is, it may be provided by being linked (eg, covalently bonded or cross-linked) to a detectable label.

The detectable label is a chromogenic enzyme (peroxidase), alkaline phosphatase, etc.), a fluorescent substance (FITC, RITC, rhodamine, TexasRed), fluorescein ), phycoerythrin, quantum dots), chromophores and radioactive isotopes ( ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ^99m Tc, ³² P, ³⁵ S, etc.) It may be at least any one selected from, but is not limited thereto.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Confirmation of aggregation state of amyloid beta 42

Amyloid beta 42 was treated with HFIP to dissolve evenly through incubation for 1 h.

Next, it was transferred to a tube and put in a hood to evaporate the highly volatile HFIP overnight. After confirming that all of the HFIP was evaporated, the HFIP was completely evaporated using a speedvac under vacuum pressure for 1 hour to obtain an amyloid beta 42 film (film).

Next, a 5 mM stock was prepared using a small amount of DMSO, followed by sonication for 10 minutes.

Finally, after dilution to 100 μM using PBST, incubated at 4 °C for 24 hours, finally diluted to 50 µg/ml using PBS, and incubated at 4 °C for each hour.

Samples incubated for each time were confirmed through a TEM image, and as a result, it was confirmed that the structure became larger and more complicated as time passed, as shown in FIG. 1 .

Example 2. Screening of peptides that specifically bind to amyloid beta 42 using phage display

To perform phage display, the amyloid beta 42 monomer obtained in Example 1 was used. A G protein-coated plate was used to immobilize the protein plate so that IgG could be directionally immobilized.

This phage selection process is a more natural protein because there is no denaturation of the protein structure compared to the method of selecting phages after fixing the protein by the hydrophobic interaction between the protein and the plate surface, which is a method mainly used in the past. The structure was shown to enable phage display with higher specificity.

As described above, the immobilized protein and random phage peptide library (phage peptide library prepared to have 2.7 billion various amino acid sequences through a random arrangement consisting of 12 linear amino acids fused to M13 phage gp3 minor coat protein-Ph.DTM, In the screening process using the Phage Display Peptide Library Kit, New England Biolabs), the experiment was designed to enable higher specificity and avidity analysis.

More specifically, in order to screen a phage peptide that binds to amyloid beta 42, an M13 phage library is put on a plate on which amyloid beta 42 is immobilized to bind with amyloid beta 42, and then binds with high affinity through various binding times and washing conditions. Peptide-expressing phages were selected.

Figure 2 shows a schematic diagram of M13 phage peptide screening for the selection of phage expressing peptides that bind to amyloid beta 42. More specifically, when amyloid beta 42 is immobilized on an IgG-coated plate, an M13 phage library in which peptides composed of random amino acids are expressed, and various washing conditions and binding time are adjusted to bind phage binding to amyloid beta 42 , it is shown in the schematic diagram that only phages that specifically bind to amyloid beta 42 can be obtained, and only phages expressing peptides that bind to amyloid beta 42 can be extracted. At 42, the M13 phage library was bound, washed under various conditions, and finally the bound phage was eluted to obtain the phage.

Amyloid beta 42 by infecting and amplifying the phage obtained by the above process with E. coli (E. coli ER2738), binding the amplified phage again with amyloid beta 42, and repeating the conditions such as adjusting the strength and reaction time of washing conditions. The process of finding phages having a higher binding affinity to It shows the biopanning conditions of 4 steps for

Round no. binding condition Binding
Time (minutes) Washing condition 1R TBS 120 0.1% TBST 2R TBS 60 0.1% TBST 3R TBS 60 0.1% TBST negative TBS + 0.5% BSA 60 4R TBS + 0.5% BSA 30 0.1% TBST, 500 mL NaCl

As shown in Table 1, the reaction conditions were variously changed with strong washing conditions and short reaction times for each step, and phages having a peptide binding to amyloid beta 42 were obtained by biopanning. After infecting the phage obtained as described above with E. coli ER2738, a host cell, and amplifying it in LB medium, about 60 phage plaques were selected and M13 phage genomic DNA (Single Strand DNA) was separated and purified to separate and purify each The gene sequence was confirmed, and the amino acid sequence of the peptide part expressed in phage surface protein (gp3 minor coat protein) and produced to bind to amyloid beta 42 was confirmed, and the sequence is as shown in Table 2 below.

linear amyloid beta 42 binding Binding Peptide order ABP #9 SEPQNIWQYLRN

Example 3. Analysis of binding force through modification and synthesis of a peptide that specifically binds to amyloid beta 42

In the peptide-expressing phage detected with high frequency, biotin is bound to indicate a signal with respect to one linear peptide (linear amyloid beta 42 binding Binding Peptide, 1ABP) sequence that has a high specific binding affinity to amyloid beta 42. synthesized. The sequence is as shown in Table 3 below.

linear amyloid beta 42 binding Binding Peptide order ABP #9 variant (Biotin)-SEPQNIWQYLRN-NH ₂

The indicated amino acid sequences are shown from the amino terminus (N-terminus). Streptavidin capable of showing a signal and biotin capable of strong binding were bound to the N-terminus, and to the C-terminus, amidation was performed to remove amino acid reactivity.

In order to analyze the binding affinity of the peptide to amyloid beta 42, the peptide was diluted by concentration on a plate on which amyloid beta 42 was fixed and bound. The absorbance of the peptide bound to amyloid beta 42 in each well of the plate was measured at 450 nm using a chromogenic reaction of HRP and TMB solution.

As a result, as shown in FIG. 4, the absorption signal according to the concentration of the peptide was labeled and the K _{d value was determined according to the saturation curve. More specifically, the binding force (K d} ) between the peptide according to the present invention and amyloid beta 42 was 17.47. Measured as ± 2.98 μM.

Example 4. Confirmation of detection range of amyloid beta 42 through quantification of amyloid beta 42 using a selected peptide probe

Anti-amyloid beta 42 was treated by serial dilution of amyloid beta 42 on a plate immobilized with IgG. The concentration of biotin-labeled peptide was treated with 100 μM and allowed to bind for 1 hour.

Next, streptavidin-HRP was diluted 1:4000 and incubated for 1 h. The TMB solution was treated for 15 minutes to induce a color reaction, and the absorbance for the amount of protein was measured at 450 nm.

As a result, as shown in FIG. 5, a section showing a linearity was set by labeling the absorbance signal according to the concentration of the target protein. More specifically, it was confirmed that the absorbance increased with the increase in the concentration of amyloid beta 42. It was confirmed that the peptide according to the invention detected amyloid beta 42 at a concentration level of ng/ml.

The experimental results of the above Examples are the results of confirming that the peptide according to the present invention can effectively detect amyloid beta 42 of a small molecule.

Example 5. Diagnosis and validation of Alzheimer's disease by measuring cerebrospinal fluid (CSF) samples

A cerebrospinal fluid (CSF) sample collected from a patient was treated on a plate immobilized with anti-amyloid beta 42 IgG. The concentration of biotin-labeled peptide was treated with 100 μM and allowed to bind for 1 hour. Streptavidin-HRP was diluted 1:4000 and incubated for 1 h. The TMB solution was treated for 15 minutes to induce a color reaction, and the absorbance for the amount of protein was measured at 450 nm.

Finally, the absorbance signal for each cerebrospinal fluid (CSF) sample was labeled and compared with the Alzheimer's disease diagnosis result through PET image.

As a result, as shown in FIG. 6 , the ELISA values of the patient group (+) in which amyloid beta 42 was accumulated in the brain and the PET signal was confirmed and the non-patient group (-) in which the PET signal was not observed were analyzed through t-test. As the p-value was calculated to be less than 0.0001, it was confirmed that when Alzheimer's disease was diagnosed using the peptide according to the present invention, meaningful classification was possible between the patient group and the non-patient group (n; (+) = 6, (-) = 7).

Example 6. Comparison of measured values of cerebrospinal fluid (CSF) samples using conventional antibody-based ELISA

A cerebrospinal fluid (CSF) sample collected from a patient was treated using a commercially available ELISA. The absorbance signal for each cerebrospinal fluid (CSF) sample was labeled and compared with the Alzheimer's disease diagnosis result through PET image.

As a result, as shown in FIG. 5, when the ELISA values of the patient group (+) in which amyloid beta 42 was accumulated in the brain and the PET signal was confirmed and the non-patient group (-) in which the PET signal was not observed were analyzed through t-test, With a p-value of 0.3470, it was confirmed that significant classification between the two patient groups was not possible (n; (+) = 6, (-) = 7).

The experimental results of Examples 5 and 6 are the results confirming that the peptide according to the present invention can diagnose Alzheimer's disease more accurately than EILSA, which is a conventional method for diagnosing Alzheimer's disease.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Industry-University Cooperation Foundation Hanyang University <120> peptide probe for detecting small molecular weight amyloid beta and use it <130> PD19-179 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> linear amyloid beta 42 binding Binding Peptide <400> 1 Ser Glu Pro Gln Asn Ile Trp Gln Tyr Leu Arg Asn 1 5 10

Claims (9)

delete delete delete A probe composition for imaging a small molecule amyloid beta protein, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
The probe composition according to claim 4, wherein the peptide is labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.
A composition for detecting low-molecular-weight amyloid beta protein, comprising the probe composition of claim 4 .
A composition for early diagnosis of Alzheimer's disease, comprising the probe composition of claim 4 .
A method for detecting a low molecular weight amyloid beta protein, comprising the steps of:
(a) contacting the peptide of claim 4 with a sample derived from a detection target; and
(b) confirming whether the peptide and amyloid beta, which may be included in the detection target-derived sample, are bound.
9. The method of claim 8,
The detection method, characterized in that the peptide is labeled with any one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.

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