WO2024076210A1 - Method for screening for candidate material for inhibiting or disaggregating amyloid protein aggregation - Google Patents

Abstract

The present invention relates to methods for screening for a drug that interferes with or modulates binding between amyloid proteins and screening for a target domain of the drug. According to a method for screening for a candidate material for inhibiting or disaggregating amyloid protein aggregation, according to an aspect, the efficacy between previously known amyloid protein aggregation inhibitors or disaggregators can be compared, target domains of amyloid protein aggregation inhibitors or disaggregators can be analyzed, and furthermore, the mechanisms thereof can be verified. Thus, the method can be effectively used in the development of an inhibitor or disaggregator that specifically disaggregates the sequence of an amyloid protein, particularly Aβ protein.

Description

Method for screening candidates for inhibiting or dissolving amyloid protein aggregation

It relates to a method for screening an amyloid protein aggregation inhibitor or disaggregator, a kit, and a method for screening the aggregation inhibition or disaggregator site of an amyloid protein aggregation inhibitor or disaggregator.

Amyloidosis is a disease that occurs when abnormal proteins called amyloid accumulate in tissues. Amyloid is a protein mass with a diameter of 7-13 nm and a beta-sheet structure that appears in a fibrous form when viewed under a microscope, and is characterized by staining with Thioflavin T (ThioT) and Congo red. there is. Amyloid is not found in the normal body, and to date, it has been reported that 36 proteins can form it (Picken, Acta Haematol. (2020), 143:322-334). Representative amyloidosis includes neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington disease, and prion disease. In addition, there are various types of amyloidosis depending on the causative protein and affected organ. There are a number of amyloidoses with different presentations.

An important pathological feature of Alzheimer's disease, one of these amyloidoses, is the formation of peptide aggregates called senile plaques, which cause synaptic dysfunction and neuronal death. One of the components of these senile plaques is amyloid-beta, which is 40-42 amino acids long. Amyloid-beta monomers are prone to self-assemble into oligomers, protofibrils, and beta-sheet-rich fibers and are associated with the pathogenesis of neurotoxicity.

Although the correlation between amyloid-beta plaques and neurotoxicity has not yet been clearly elucidated, the self-assembly of amyloid-beta into intermediate oligomers or aggregates is believed to be involved in the pathogenesis of cranial nerve diseases such as Alzheimer's disease.

However, the majority of amyloid disease treatments developed to date use antibodies against Aβ fragments or specific epitopes within Aβ that cause an immune response in patients, or apply chemical drugs. In the case of chemical drugs, there is currently no fundamental treatment for amyloid disease.

Accordingly, the present inventors completed the present invention to provide a method of screening for a therapeutic agent for degenerative brain diseases that inhibits the formation of amyloid protein, especially amyloid beta plaques.

One aspect is to provide a method of screening for substances that inhibit, inhibit, or dissolve the aggregation of amyloid proteins.

Another aspect is to provide a kit for screening substances that inhibit, inhibit, or dissolve the aggregation of amyloid proteins.

Another aspect is to provide a method of screening for an aggregation inhibition or dissolution site of action of a substance that inhibits, inhibits, or dissolves the aggregation of amyloid protein.

The present invention provides a method for screening for an aggregation inhibitor or an aggregation-dissolving agent of amyloid protein, for example, an amyloid protein aggregation inhibitor or Methods and kits for screening candidate solubilizers are provided.

One aspect includes (1) providing a plate to which a first amyloid protein is attached;

(2-1) contacting the plate with a second amyloid protein bound to a protein aggregation inhibitor candidate and an indicator; or (2-2) contacting a second amyloid protein to which an indicator substance is bound to the plate and then treating the candidate substance as a protein aggregation dissolving agent; And, (3-1) measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-1) and selecting an aggregation inhibitor candidate changed compared to the untreated control group; or (3-2) measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-2) and selecting an aggregation resolving agent candidate changed compared to the untreated control group. A method for screening solubilizers is provided.

In one embodiment, step (2-1) or (2-2) may further include inducing aggregation of the first and second amyloid proteins.

In one embodiment, the method of screening for the protein aggregation inhibitor or aggregation dissolving agent is an amyloid protein aggregation inhibitor or a candidate material that changes the aggregation rate of amyloid protein compared to the substance that changed the aggregation rate of the amyloid protein or the untreated control. It may further include a step of selecting as a coagulant dissolving agent.

As used herein, “amyloid protein” refers to a protein that tangles and aggregates in the form of a beta plate structure, and refers to a protein or a fragment thereof capable of forming amyloid deposits. In the present invention, amyloid protein may refer to individual proteins constituting amyloid protein aggregates.

In one embodiment, the amyloid protein may be amyloid beta, tau protein, alpha-synuclein, prion, amyloidogenic protein, amyloid transthyretin, Lewy body, fliglutamine, Torsin A, and AIMP2 protein.

As used herein, the term “amyloid beta” is a peptide molecule containing 36 to 43 amino acids, is a major component of amyloid plaques, and is known to be involved in the development of Alzheimer's disease. The amyloid beta peptide molecules aggregate to form neurotoxic oligomers, causing degenerative brain diseases. The amyloid beta peptide molecule may be obtained by chopping amyloid precursor protein (APP); UniProtKB P05067) with beta secretase and gamma secretase.

In one embodiment, the amyloid protein may self-assemble.

As used herein, the term “amyloid protein aggregate” refers to amyloid protein aggregates that are associated with disease states. These disease states include, but are not limited to, cell death, or partial or complete loss of neuronal signaling between two or more cells. Amyloid protein aggregates may be located inside the cell or outside the cell. Specifically, amyloid protein aggregates can cause neurotoxicity and cause degenerative brain diseases described later.

In one embodiment, the amyloid protein aggregates may include polymers of amyloid proteins, fibril precursors, fibrils, plaques, protein monomers and polymers bound to homologous proteins. The fibrils may include oligomers of amyloid protein. Specifically, the self-assembling protein may be soluble amyloid beta aggregates and/or water-insoluble amyloid beta aggregates, amyloid beta oligomers, amyloid beta protofibrils, amyloid beta fibrils, and amyloid beta plaques. You can.

In one embodiment, the first or second amyloid protein may be a full-length protein and/or a protein fragment.

In one embodiment, the full-length protein may be an amyloid beta _1-36, amyloid beta _1-38, amyloid beta _1-40, or amyloid beta _1-42 peptide.

As used herein, the term “protein fragment” refers to a truncated form of a protein. Specifically, the protein fragment refers to a cleaved portion of the full-length protein.

In one embodiment, the protein fragment may be a continuous protein within an amyloid protein. Specifically, it may be a continuous 6 to 15 mer within the amyloid protein.

In one embodiment, the protein fragment may be 6 to 15 mer.

In one embodiment, the protein fragment may be 6 to 14 mer. In one embodiment, the protein fragment may be 6 to 13 mer. In one embodiment, the protein fragment may be 6 to 12 mer. In one embodiment, the protein fragment may be 6 to 11 mer. In one embodiment, the protein fragment may be 6 to 10 mer. In one embodiment, the protein fragment may be 6 to 9 mer.

In one embodiment, the protein fragment may be amyloid beta 6mer (hereinafter also referred to as amyloid beta hexamer).

The protein fragment may be DAEFRH. The protein fragment may be AEFRHD. The protein fragment may be EFRHDS. The protein fragment may be FRHDSG. The protein fragment may be RHDSGY. The protein fragment may be HDSGYE. The protein fragment may be DSGYEV. The protein fragment may be SGYEVH. The protein fragment may be GYEVHH. The protein fragment may be YEVHHQ. The protein fragment may be EVHHQK. The protein fragment may be VHHQKL. The protein fragment may be HHQKLV. The protein fragment may be HQKLVF. The protein fragment may be QKLVFF. The protein fragment may be KLVFFA. The protein fragment may be LVFFAE. The protein fragment may be VFFAED. The protein fragment may be FFAEDV. The protein fragment may be FAEDVG. The protein fragment may be AEDVGS. The protein fragment may be EDVGSN. The protein fragment may be DVGSNK. The protein fragment may be DVGSNG. The protein fragment may be VGSNGA. The protein fragment may be GSNGAI. The protein fragment may be SNGAII. The protein fragment may be NGAIIG. The protein fragment may be GAIIGL. The protein fragment may be AIIGLM. The protein fragment may be IIGLMV. The protein fragment may be IGLMVG. The protein fragment may be GLMVGG. The protein fragment may be LMVGGV. The protein fragment may be MVGGVV. The protein fragment may be VGGVVI. The protein fragment may be GGVVIA.

In one embodiment, in the step of providing a plate to which the first amyloid protein is attached, the first amyloid protein may be attached to the plate by cysteine bound to the C-terminus. Specifically, the full-length protein amyloid beta _1-42 or amyloid beta hexamer may be attached to the plate through a cysteine bound to the C-terminus.

In one embodiment, the cysteine may be bound to the C-terminus of the first amyloid protein through systemic anhydride activation, but is not limited thereto. Binding of cysteine to the C- or N-terminus of amyloid protein can be performed by methods that are obvious to those skilled in the art.

In one embodiment, the peptide having cysteine bound to the C-terminus of the amyloid protein may be synthesized through a solid-phase peptide synthesis method. Specifically, it may be by synthesizing 9-fluorenylmethyloxycarbonyl solid phase peptide using microwaves, but is not limited thereto.

In one embodiment, full-length amyloid beta _1-42 or amyloid beta hexamer with cysteine bound to the C-terminus is formed by binding cysteine to the C-terminus through systemic anhydride activation, and then activating it in the microwave. It can be produced by 9-fluorenylmethyloxycarbonyl solid-phase peptide synthesis using .

In one embodiment, the full-length amyloid beta _1-42 or amyloid beta hexamer with cysteine bound to the C-terminus may be attached to the C-terminus by reacting cysteine with a functional group of the plate, specifically maleimide. and its derivatives, aziridine and its derivatives, acryloyl and its derivatives, or aryl halide and its derivatives.

In one embodiment, the first amyloid protein may be attached to the plate in monomeric form and may be attached to the plate to maintain the monomeric form before forming aggregates by contact with the second amyloid protein.

In one embodiment, the method includes the steps of (1) providing a plate to which full-length amyloid beta _1-42 or amyloid beta hexamer is attached through a cysteine bound to the C terminus; (2-1) contacting the plate with full-length amyloid beta _1-42 to which a protein aggregation inhibitor candidate and an indicator are bound, or (2-2) contacting full-length amyloid beta _1-42 to which an indicator is bound. Afterwards, processing the protein aggregation dissolving candidate material; And, (3-1) selecting an aggregation inhibitor candidate that inhibits aggregation between amyloid beta livers compared to the untreated control group; Alternatively, a method of screening a protein aggregation inhibitor or an aggregation-dissolving agent is provided, which includes a step of selecting a candidate for an aggregation-dissolving agent that dissolves aggregation between aggregated amyloid beta compared to an untreated control group.

The method may further include measuring the aggregation rate between amyloid beta.

In one embodiment, the step of contacting the second amyloid protein may further include inducing aggregation with the first amyloid protein.

In one embodiment, the second amyloid protein may be bound to an indicator substance.

The indicator material is for measuring the degree of binding between the first amyloid protein and the second amyloid protein; And it may be used to measure the aggregation inhibition or aggregation dissolution ability of the first amyloid protein and the second amyloid protein by the aggregation inhibitor, aggregation dissolving agent, aggregation inhibitor candidate, or aggregation dissolving agent candidate, specifically, the degree of aggregation inhibition or aggregation dissolution. there is.

In one embodiment, the indicator may be a fluorescent substance or a fluorescent protein fragment.

In one embodiment, the fluorescent material is a fluorescent dye having rhodamine, coumarin, EvoBlue, oxazine, carbopyronine, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, or carbazole as a basic skeleton, or It may be a derivative of the fluorescent dye.

Specifically, the fluorescent substance is Fluorescein, CR110:Carboxyrhodamine 110:Rhodamine Green (trade name), TAMRA:Carboxytetramethylrhodamine:TMR, Carboxyrhodamine 6G:CR6G, BODIPY FL (trade name): 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-difluoro- 1,3,5,7-Tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G (trade name): 4,4-difluoro-5-(4- Phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-difluoro-5-( 2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-difluoro-5-styryl-4-bora -3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a- Diaza-s-indacene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid, EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 655 (trade name), ATTO 680 (trade name), ATTO 700 (trade name), ATTO MB2 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name), Alexa Fluor633 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor790 (trade name), Flamma 496 (trade name), Flamma 507 (brand name), Flamma 530 (brand name), Flamma 552 (brand name), Flamma560 (brand name), Flamma 575 (brand name), Flamma 581 (brand name), Flamma 648 (brand name), Flamma 675 (brand name), Flamma749 (brand name) ), Flamma 774 (trade name), Flamma 775 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name), 5(6)-TAMRA-X (trade name), 5TAMRA (trade name), Indocyanine green ( ICG) and 2-((E)-2-((E)-2-(4-(2-carboxyethyl)phenoxy)-3-((E)-2-(3,3-dimethyl-5-sulfonato- 1-(3-(tri-methyl ammonio)-propyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-(trimethyl ammonio)-propyl)- It may be selected from the group consisting of 3H-indolium-5-sulfonate disodium bromide (ZW800-1).

In one embodiment, the fluorescent protein fragments include Venus, Cerulean, Citrine, and mKate, and may be fluorescent proteins of different colors, or may be parts thereof.

As used herein, the term “amyloid protein aggregation inhibitor” refers to inhibiting the accumulation of amyloid proteins, and the term “amyloid protein aggregation dissolving agent” may refer to a substance that decomposes the aggregation of already formed amyloid proteins. The aggregation inhibitor or aggregation dissolving agent may refer to a substance that inhibits self-assembly of amyloid proteins or breaks down aggregation of already formed self-assembled proteins. Specifically, it may refer to a substance that inhibits aggregation or disaggregates water-soluble protein aggregates and/or water-insoluble protein aggregates.

In one embodiment, the aggregation inhibitor or aggregation dissolving agent may inhibit or dissolve the aggregation of amyloid beta, tau protein, or alpha-synuclein.

In one embodiment, the aggregation inhibitor inhibits amyloid fibrillation and amyloid fiber aggregation, and the aggregation dissolving agent includes the dissociation of aggregates. Specifically, it dissolves or accumulates soluble amyloid beta aggregates and/or water-insoluble amyloid beta aggregates, amyloid beta oligomers, amyloid beta protofibrils, amyloid beta fibrils, and amyloid beta plaques. It is an inhibitory substance.

In one embodiment, the amyloid protein aggregation inhibitor or aggregation-dissolving agent may be a treatment for degenerative brain disease.

In one embodiment, the aggregation inhibitor or aggregation inhibitor candidate may be treated simultaneously or sequentially with the second amyloid protein.

In one embodiment, the aggregation inhibitor may be treated simultaneously with the second amyloid protein. Specifically, the amyloid beta aggregation inhibitor curcumin or scyllo-inositol may be treated simultaneously with fluor-Aβ _1-42 .

In one embodiment, the aggregation-dissolving agent or aggregation-dissolving agent candidate may be treated after aggregating the first amyloid protein and the second amyloid protein.

Specifically, the amyloid beta aggregation dissolving agent necrostatin-1 and sunitnib may be treated sequentially with fluor-Aβ _1-42 , for example, after 24 hours.

The protein aggregation inhibitor or aggregation-dissolving agent is used in the prevention and treatment of degenerative brain diseases such as (1) accumulation of amyloid beta, (2) aging of brain cells, (3) loss of synapses, and (4) accumulation of peripheral immune cells. It may alleviate the pathological symptoms of degenerative brain disease, specifically Alzheimer's disease.

As used herein, the term “degenerative brain disease” refers to all diseases related to degenerative changes in the brain, especially all diseases (brain diseases) that can be caused by factors such as aggregation of amyloid-beta in the brain and/or brain nerve cells. It is used to describe comprehensively. In one embodiment, the degenerative brain disease includes dementia, Alzheimer's disease, preclinical Alzheimer's disease, Parkinson's disease, Huntington's disease, and mild cognitive impairment. , cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, Spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia ), progressive supranuclear palsy, and frontotemporal dementia. Preferably, the degenerative brain disease may be Alzheimer's disease.

As used herein, the term "Alzheimer's disease" is used interchangeably with senile dementia and refers to a disease involving mental deterioration associated with a specific degenerative brain disease characterized by senile plaques, neuroinflammatory tangles, and progressive nerve loss. You can.

As used herein, the term "Parkinson's disease" may refer to a chronic and progressive degenerative disease of the central nervous system that often impairs motor function and language ability.

As used herein, the term "Huntington's disease" may refer to a neurodegenerative disease that is caused by a 3-base repeat expansion in the gene encoding the huntingtin protein and is accompanied by chorea, psychosis, dementia, etc.

As used herein, the term "Lou Gehrig's disease" refers to a disease in which only motor neurons are selectively killed, and may refer to a disease in which both upper motor neurons in the cerebral cortex and lower motor neurons in the brainstem and spinal cord are gradually destroyed.

As used herein, the term “Pick's disease” may refer to a disease that causes progressive destruction of nerve cells in the brain.

The term "tauopathy" of the present invention may refer to a neurodegenerative disease in which cranial nerves are damaged due to abnormal accumulation of tau protein (a family closely related to intracellular microtubul-related proteins) in brain tissue.

In one embodiment, the method includes providing a plate to which a first amyloid protein is attached with cysteine bound to the C-terminus;

(2-1) contacting the plate with a second amyloid protein to which a protein aggregation inhibitor candidate and an indicator are bound, or (2-2) contacting a second amyloid protein to which an indicator is bound, and then protein aggregation. Processing the solubilizer candidate; And, (3-1) selecting an aggregation inhibitor candidate that inhibits aggregation between the first amyloid protein and the second amyloid protein compared to the untreated control group; Alternatively, a method for screening a protein aggregation inhibitor or an aggregation dissolving agent comprising the step of selecting a candidate for an aggregation dissolving agent that dissolves the aggregation between the first amyloid protein and the second amyloid protein compared to the untreated control group is provided.

The method may further include measuring the aggregation rate between the first amyloid protein and the second amyloid protein.

In one embodiment, the step of contacting the second amyloid protein may further include inducing aggregation with the first amyloid protein.

Specifically, the full-length protein amyloid beta _1-42 or the protein fragment amyloid beta hexamer may be attached to the plate with cysteine bound to the C-terminus.

In the step of providing the plate to which the first amyloid protein is attached, the concentration of the first amyloid protein treated on the plate may be 0.00001 μM to 50 μM. In one embodiment, the concentration of the first amyloid protein may be 0.01 μM to 20 μM. In one embodiment, the concentration of the first amyloid protein may be 0.1 μM to 15 μM. In one embodiment, the concentration of the first amyloid protein may be 0.1 μM to 10 μM. In one embodiment, the concentration of the first amyloid protein may be 0.5 μM to 5 μM. Specifically, the concentration of the first amyloid protein may be 1 μM. If the concentration of the first amyloid protein processed on the plate is 0.1 μM or less, the fixation effect of the first amyloid protein fixed on the plate is low, making it difficult to use it in the screening method of the present invention. If the concentration exceeds 10 μM, the fixation effect of the first amyloid protein immobilized on the plate is saturated, and the concentration of the first amyloid protein present on the plate without being fixed due to non-specific binding increases, causing an error in measuring the aggregation rate. As it can be produced, the concentration of the first amyloid protein of the present invention should be 0.1 μM to 10 μM, preferably 0.5 μM to 5 μM.

In one embodiment, in the step of providing a plate to which the first amyloid protein is attached, the first amyloid protein may be attached to the plate by treating the plate and incubating at 20°C to 40°C. In one embodiment, the amyloid protein may be attached to the plate by incubation at 25 to 35°C. Specifically, the amyloid protein may be attached to the plate by incubation at 28 to 32°C. When the first amyloid protein is attached by incubating at less than 25°C or more than 35°C, the attachment efficiency to the plate is significantly low and cannot be applied to the screening method for aggregation inhibitor or aggregation dissolving agent. Therefore, the first amyloid protein is treated on the plate. It should be adhered to the plate by incubation at 25°C to 35°C, preferably 28 to 32°C.

In one embodiment, in the step of providing a plate to which the first amyloid protein is attached, the first amyloid protein may be attached to the plate by treating the plate and incubating for 12 to 36 hours. In one embodiment, the amyloid protein may be attached to the plate by incubating for 20 to 26 hours. Specifically, the first amyloid protein may be attached to the plate by incubating for 23 to 25 hours. When the amyloid protein is incubated for less than 20 hours, the fixation effect of the first amyloid protein immobilized on the plate is low, making it difficult to use it in the screening method of the present invention, and when the amyloid protein is incubated for more than 26 hours, the fixation effect is saturated, so the experiment In terms of speed and business feasibility, the first amyloid protein should be attached to the plate by incubating for 20 to 26 hours, preferably 23 to 25 hours.

In one embodiment, in providing a plate to which the first amyloid protein is attached, the first amyloid protein may be attached to the plate at pH 4 to 9. In one embodiment, the first amyloid protein may be attached to the plate at pH 5 to 8. Specifically, the first amyloid protein may be attached to the plate at pH 6.5 to 7.2. When the first amyloid protein is attached to the plate at pH less than 5 or more than pH 8, the fixation effect of the first amyloid protein is low and the screening efficiency is reduced when used in the screening method of the present invention. Therefore, the first amyloid protein is used at pH 5 or higher. It should adhere to the plate at pH 8, preferably pH 6.5 to 7.2.

In one embodiment, in the step of contacting the second amyloid protein with the plate to which the first amyloid protein is attached, the second amyloid protein is treated at a concentration in the range of 1 μM to 15 μM to the plate to which the first amyloid protein is attached. It can be. In one embodiment, it can be treated at a concentration ranging from 5 μM to 15 μM. Specifically, it can be treated at a concentration within the range of 8 μM to 12 μM. When the second amyloid protein is treated at less than 5 μM, the aggregation rate between the first and second amyloid proteins is low, making it difficult to use in the screening method of the present invention, and when the second amyloid protein is treated at more than 15 μM, non-specific binding occurs. The concentration of the second amyloid protein that does not aggregate with the first amyloid protein and is present on the plate increases, which may cause errors in measuring the aggregation rate. The second amyloid protein is added to the plate to which the first amyloid protein is attached at 5 μM to 15 μM. , preferably at 8 μM to 12 μM.

In one embodiment, in the step of contacting the second amyloid protein with the plate to which the first amyloid protein is attached, the second amyloid protein is treated to the plate to which the first amyloid protein is attached and incubated at 20°C to 40°C. By incubation, it can be aggregated with the first amyloid protein. In one embodiment, the amyloid protein can be aggregated with the first amyloid protein by incubating at 28 to 38°C. Specifically, the amyloid protein may be incubated at 30 to 36° C. to aggregate with the first amyloid protein. When the first amyloid protein is incubated at less than 28°C or more than 38°C to cause aggregation with the first amyloid protein, the efficiency of aggregation with the first amyloid protein is significantly low and cannot be applied to the screening method for an aggregation inhibitor or an aggregation dissolving agent. 2 The amyloid protein should be treated on a plate to which the first amyloid protein is attached and incubated at 28 to 38°C, preferably 30 to 36°C to aggregate with the first amyloid protein.

In one embodiment, in the step of contacting the second amyloid protein with the plate to which the first amyloid protein is attached, the second amyloid protein can be brought into contact with the plate to which the first amyloid protein is attached at pH 4 to 9. there is. In one embodiment, the second amyloid protein may be brought into contact with a plate to which the first amyloid protein is attached at pH 5 to 8. Specifically, the second amyloid protein can be brought into contact with the plate to which the first amyloid protein is attached at pH 7 to 8. When the second amyloid protein is brought into contact with the plate to which the first amyloid protein is attached at a pH of less than 5 or more than pH 8, the aggregation effect with the first amyloid protein is low, making it difficult to use the second amyloid protein in the screening method of the present invention. The amyloid protein should be brought into contact with the plate to which the first amyloid protein is attached at pH 5 to pH 8, preferably pH 7 to pH 8.

Another aspect includes a plate to which a first amyloid protein is attached; and a kit for screening a protein aggregation inhibitor or aggregation dissolving agent containing a second amyloid protein to which an indicator substance is bound.

In one embodiment, the first and second amyloid proteins may be full-length proteins or protein fragments.

In one embodiment, the first and second amyloid proteins may be self-assembled.

The “self-assembling protein,” “protein fragment,” “aggregation inhibitor,” and “aggregation dissolving agent” are as described above.

In one embodiment, the aggregation inhibitor may be treated simultaneously or sequentially with the second amyloid protein.

In one embodiment, the aggregation dissolving agent may be used after aggregating the first amyloid protein and the second amyloid protein.

The present invention provides a method for searching the action site of an amyloid protein aggregation inhibitor or aggregation dissolving agent using amyloid protein and fragments thereof, for example, in subjects with degenerative brain disease or at risk of developing degenerative brain disease, Provides a method for searching the action site (specifically, the action domain) of an amyloid protein aggregation inhibitor or aggregation dissolving agent to treat or prevent.

Another aspect includes (1) providing a plate to which a plurality of first amyloid protein fragments are attached; (2-1) contacting the plate with a second amyloid protein bound to a protein aggregation inhibitor candidate and an indicator; or (2-2) contacting the plate with a second amyloid protein bound to an indicator material and then treating the candidate material as a protein aggregation dissolving agent; (3-1) measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-1); or (3-2) screening the site where the amyloid protein aggregation inhibitor candidate or aggregation dissolving agent candidate acts, including the step of measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-2). Provides a way to do this.

The method may additionally include sorting the degree of inhibition or dissolution of aggregation between the first amyloid fragment and the second amyloid protein by the first amyloid protein fragment.

In one embodiment, step (2-1) or (2-2) may further include inducing aggregation of the first and second amyloid proteins.

In one embodiment, the amyloid protein may be amyloid beta, tau protein, or alpha-synuclein.

In one embodiment, the protein fragment is a contiguous 6 to 15 mer within an amyloid protein, and the plurality of protein fragments may include two or more overlapping contiguous amino acid residues. The step of measuring the aggregation rate may be done by analyzing the intensity of the indicator material and comparing it for each protein fragment.

In one embodiment, the method may further include determining a protein fragment for which a high degree of aggregation inhibition or aggregation dissolution is measured as the site of action of the candidate substance. Specifically, one protein fragment for which a high degree of aggregation inhibition or aggregation dissolution is measured is determined as the action site, or an overlapping amino acid residue of a plurality of protein fragments for which aggregation inhibition or aggregation dissolution is measured to be high is determined as the action site. It may include more steps. More specifically, the step of determining a protein fragment as an action site is selecting a protein fragment that shows a higher value when comparing the aggregation inhibition rate (%) or aggregation dissolution rate (%) for each protein fragment through the method shown in the examples. It may include.

In one embodiment, the action site may be present in two or more amyloid proteins.

The “self-assembling protein,” “protein fragment,” “aggregation inhibitor,” and “aggregation dissolving agent” are the same as described above.

In one embodiment, the amyloid protein may be amyloid beta, tau protein, or alpha-synuclein.

In one embodiment, the protein fragment may be 6 to 15 mer. Specifically, the protein fragment may be a 6mer.

In one embodiment, the aggregation inhibitor or aggregation inhibitor candidate may be treated simultaneously or sequentially with the second amyloid protein.

In one embodiment, the aggregation-dissolving agent or aggregation-dissolving agent candidate may be treated after aggregating the first amyloid protein and the second amyloid protein.

In one embodiment, the method of screening the site where the amyloid protein aggregation inhibitor candidate or aggregation dissolving agent candidate acts is a molecular docking simulation used to predict and analyze the target site of the Aβ aggregation inhibitor or dissolving agent. (molecular docking simulation) can not only be used to verify research results, but can also be used to develop aggregation inhibitors or solubilizers that specifically inhibit or release aggregation in the Aβ sequence using a rational design method.

According to an aspect of the screening method for candidates for inhibiting or dissolving amyloid protein aggregation, not only can the efficacy of existing known amyloid protein aggregation inhibitors or solubilizers be compared, but also the target domains of amyloid protein aggregation inhibitors or solubilizers are analyzed, and further the mechanism is determined. Since it can reveal the identity of amyloid proteins, especially Aβ, it can be useful in developing inhibitors or solubilizers that dissolve the aggregation of specific protein sequences.

Figure 1 is a schematic diagram illustrating a method for producing amyloid beta and amyloid beta fragments with cysteine attached to the C-terminus.

Figure 2 is a schematic diagram showing the types of amyloid hexamer fragments with cysteine attached to the C-terminus.

Figure 3 is a graph showing the results of analysis of hexameric fragments of amyloid beta _1-6 to amyloid beta _10-15 by reverse phase-HPLC.

Figure 4 is a graph showing the results of analysis of hexameric fragments of amyloid beta _11-16 to amyloid beta _20-25 by reverse phase-HPLC.

Figure 5 is a graph showing the results of analysis of hexameric fragments of amyloid beta _21-26 to amyloid beta _30-35 by reverse phase-HPLC.

Figure 6 is a graph showing the results of reverse phase-HPLC analysis of hexameric fragments of amyloid beta _31-36 to amyloid beta _37-42 and amyloid beta _1-42 .

Figure 7 is a graph confirming the optimal conditions (peptide concentration, fixation time, temperature, pH, and fluorescent label concentration) of A3 and MAP.

Figure 8 shows the results of confirming structural, morphological, and cohesive changes in A3 and MAP peptides.

Figure 9 is a schematic diagram showing a method for screening candidate substances as amyloid beta aggregation inhibitors.

Figure 10 is a schematic diagram showing a method for screening candidate substances as an amyloid beta aggregation dissociator.

Figure 11 is a graph confirming the aggregation inhibition effect of amyloid beta aggregation inhibitors (Curcumin and Scyllol-inositol) using A3.

Figure 12 is a graph confirming the aggregation-dissolving effect of amyloid beta aggregation-dissolving agents (Necrostantin-1 and Sunitinib) using A3.

Figure 13 is a schematic diagram showing a method for screening candidate substances as amyloid beta aggregation inhibitors.

Figure 14 is a schematic diagram showing a method for screening candidate substances as an amyloid beta aggregation dissociator.

Figure 15 is a graph confirming the aggregation-dissolving action site of the amyloid beta aggregation inhibitor (Curcumin) using MAP.

Figure 16 is a graph confirming the aggregation inhibition site of amyloid beta aggregation inhibitor (Scyllol-inositol) using MAP.

Figure 17 is a graph confirming the aggregation-dissolving action site of amyloid beta aggregation-dissolving agent (Necrostantin-1) using MAP.

Figure 18 is a graph confirming the aggregation inhibitory action site of amyloid beta aggregation dissolving agent (Sunitinib) using MAP.

Figure 19 is a schematic diagram showing the A3 manufacturing method.

Figure 20 is a schematic diagram showing the MAP manufacturing method.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and the content of the present invention is not limited by the following examples. The embodiments may be subject to various changes, and the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

Example 1. Production of A3 and MAP

1.1. C-terminal cysteine-conjugated amyloid beta synthesis

For A3 (Aβ aggregation assay) and MAP (mapping amyloid plate) production, Aβ _1-42 hexamer fragment with 37 C-terminal cysteines attached and full-length Aβ _1-42 with C-terminal cysteine attached were separated using microwaves. -Fluorenylmethyloxycarbonyl was synthesized through a solid phase peptide synthesis method, and is shown in Figure 2.

Specifically, the first cysteine at the C-terminus was attached using symmetric anhydride activation. 2.2 mmol of Fmoc-Cys(trt)-OH, 1.0 mmol of DIC, and 51 mg of DMAP were dissolved in 2 mL of DMF/DCM (1:1, v/v) solution, and then 0.25 mmol of Wang's solution swollen in DMF. It was reacted with resin LS for 1.5 hours. The remaining protein sequences were sequentially coupled using an automated peptide synthesizer. After synthesis, cleavage cocktail (92.5:2.5:2.5:2.5 TFA/deionized water/TIS/DODT, v/v/v/v) was added and reacted for 4 hours. TFA was evaporated using a rotary evaporator, cooled anhydrous ether was added, and centrifuged for 15 minutes to produce an unrefined peptide, which is shown in Figure 1.

The Aβ _1-42 hexamer fragment with 37 C-terminal cysteines attached was purified and analyzed using reversed-HPLC, and is shown in Figures 3 to 6.

Figure 1 is a schematic diagram illustrating a method for producing amyloid beta and amyloid beta fragments with cysteine attached to the C-terminus.

Figure 2 is a schematic diagram showing the types of amyloid hexamer fragments with cysteine attached to the C-terminus.

Figures 3 to 6 are graphs showing the results of analysis of amyloid beta hexamer fragments by reversed phase-HPLC.

1.2. Production of A3

To prepare A3, 200 μL of washing buffer (0.1 M Na ₃ PO ₄ , 0.15 M NaCl, 0.05% Tween 20, pH 7.2) was added to the maleimide-attached plate, and washing was performed. The full-length Aβ _1-42 -Cys solution was mixed (50 μg/mL, 5% DMSO) in binding buffer (0.1 M Na ₃ PO ₄ , 0.15 M NaCl, 10 mM EDTA). 100 μL of aqueous peptide solution (10 μM) was added to each well (peptide well) and incubated at 24°C for 24 hours. 200 μL of washing buffer was added and washing was performed. The cysteine solution was mixed with binding buffer (10 μg/mL). 200 μL of the cysteine solution (10 μg/mL) mixed with the binding buffer was added to each well (blank well) and incubated at 24°C for 1 hour. Washing was performed by adding 200 μL of washing buffer, which is shown in Figure 19.

Figure 19 is a schematic diagram showing the A3 manufacturing method.

1.3. Creation of MAP

To prepare MAP, 200 μL of washing buffer (0.1 M Na ₃ PO ₄ , 0.15 M NaCl, 0.05% Tween-20, pH 7.2) was added to the maleimide-attached plate and washed. Each of the 37 Aβ _1-42 fragment-Cys solutions prepared in 1.1 above was mixed (50 μg/mL, 5% DMSO) in binding buffer (0.1 M Na ₃ PO ₄ , 0.15 M NaCl, 10 mM EDTA), and the full length Aβ _1-42 -Cys solution and binding buffer were mixed (50 μg/mL, 5% DMSO). 100 μL of aqueous peptide solution (10 μM) was added to each well (peptide well) and incubated at 24°C for 24 hours. 200 μL of washing buffer was added and washing was performed. The cysteine solution was mixed with binding buffer (10 μg/mL). 200 μL of the cysteine solution (10 μg/mL) mixed with the binding buffer was added to each well (blank well) and incubated at 24°C for 1 hour. 200 μL of washing buffer was added and washing was performed, which is shown in Figure 20.

Figure 20 is a schematic diagram showing the MAP manufacturing method.

Experimental Example 1. Optimization conditions of A3/MAP plate

1.1. Establishing optimal conditions for screening aggregation inhibitors or aggregation dissolving agents

To find the optimal conditions for the production and use of A3/MAP, experiments were conducted on peptide fixation concentration, peptide fixation time, peptide fixation temperature, peptide fixation pH, and the pH and temperature of fluor-Aβ _1-42 .

In all experiments, the amount of Aβ _1-42 peptide on the plate was indirectly compared and analyzed using anti-Aβ monoclonal 6E10 antibody, which detects Aβ _1-42 peptide. Specifically, experiments were conducted under eight concentration conditions (0, 0.00001, 0.0001, 0.001, 0,01, 0.1, 1, 10 μM) to determine the optimal peptide concentration for immobilizing the purified peptide of Example 1 on the plate. To determine the optimal time for immobilizing the purified peptide on the plate, the process was performed under 10 time conditions (0, 4, 8, 12, 16, 20, 24, 28, 32, 36 h). Experiments were conducted under 6 temperature conditions (0, 4, 24, 34, 54, 74°C) to determine the optimal temperature for immobilizing the purified peptides on the plate, and 7 conditions were used to determine the optimal temperature for immobilizing the purified peptides on the plate. The experiment was conducted under pH conditions (1.2, 3.2, 5.2, 7.2, 9.2, 11.2, 13.2). In addition, since A3/MAP plates are treated with Aβ1-42 (fluor-Aβ1-42) peptide bound to a fluorescent substance to cause Aβ aggregation, in order to find the optimal conditions for Aβ aggregation to occur in the wells of the plate, three methods were used. The experiment was conducted under fluor-Aβ _1-42 concentration conditions (0.1, 1, 10 μM), and fluor-Aβ _1-42 peptide was incubated at a concentration of 10 μM for 24 hours under seven pH conditions (1.2, 3.2, 5.2, 7.2, 9.2, 11.2, 13.2), and to find the optimal conditions for generating Aβ aggregation in the wells of the plate, fluor-Aβ _1-42 peptide was used at a concentration of 10 μM for 24 hours under six temperature conditions (0, 4 , 24, 34, 54, 74°C). The results are shown in Figure 7.

Figure 7 is a graph confirming the optimal conditions (peptide concentration, fixation time, temperature, pH, and fluorescent label concentration) of A3 and MAP.

As shown in Figure 7, with respect to the peptide concentration conditions for immobilizing the purified peptide on the plate, it was confirmed that the Aβ peptide was bound to the plate in a concentration-dependent manner. However, it was observed that the concentration no longer increased in a concentration-dependent manner above 1 μM. Through this, it was confirmed that 1 μM concentration of purified peptide was the optimal concentration for immobilizing the purified peptide on the plate (Figure 7A). Regarding the peptide time conditions for immobilizing the purified peptides on the plate, it was confirmed that each peptide was effectively immobilized on the plate for 23-36 hours using the 6E10 antibody at a concentration of 1 μM. However, it was confirmed that no additional Aβ peptide was fixed after 25 hours of incubation (Figure 7B). Regarding the peptide temperature conditions for immobilizing the purified peptides on the plate, each peptide was treated on the plate at a concentration of 1 μM, and as confirmed with the 6E10 antibody, the efficiency of immobilizing the peptides on the plate was significantly lower when the temperature was higher than 35°C. It was confirmed that he lost. Through this, it was confirmed that the fixation temperature was most effective in the range of 28-32°C (Figure 7C). Regarding the peptide pH conditions for immobilizing the purified peptides on the plate, each peptide was treated on the plate at a concentration of 1 μM and confirmed with the 6E10 antibody, and the results showed that the peptide was effectively attached to the plate in the neutral pH range, especially at pH 6.5- At 7.2, it was confirmed that the peptide was most effectively attached to the plate (Figure 7D).

Regarding the optimal conditions for generating aggregation between fixed Aβ _1-42 and fluor-Aβ _1-42 on the plate, regarding the concentration condition of fluor-Aβ _1-42 , 0.1 when comparing the intensity of fluorescence with the blank well. At a concentration of 1 μM, aggregation between fixed Aβ _1-42 and fluor-Aβ _1-42 was not well formed, but at a concentration of 10 μM, significant aggregation occurred and fluorescence intensity was detected (Figure 7G). In addition, as a result of inducing aggregation of fluor-Aβ _1-42 with fixed Aβ _1-42 at a concentration of 10 μM for 24 hours under various pH and temperature conditions, the result showed that the aggregation was most effective when the pH was in the neutral range, especially when pH was 7-8. Aggregation between effectively immobilized Aβ _1-42 and fluor-Aβ _1-42 was confirmed (Figure 7E). In addition, regarding the optimal conditions for generating aggregation between immobilized Aβ _1-42 and fluor-Aβ _1-42 on a plate, the temperature of immobilized Aβ 1-42 and fluor-Aβ _1-42 was most effective at a temperature of 28-38°C, especially 30-36°C. Aggregation between fluor-Aβ _1-42 was confirmed.

From the above results, the optimal conditions for immobilizing the purified peptide on the plate are to immobilize the peptide at a concentration of 0.5 to 5 μM at 28-32°C and pH 6.5-7.2 for 23-25 hours, and then apply a fluorescent label to induce aggregation. It was confirmed that peptides can most effectively induce aggregation with purified peptides under the conditions of 8 to 12 μM, pH 7-8, and 30-36°C temperature.

1.2. Structural/conformational/aggregative changes in peptides

Using anti-Aβ monoclonal 6E10 antibody and anti-oligomer polyclonal A11 antibody, it was confirmed whether the full-length Aβ _1-42 immobilized on the plate maintained a stable monomeric state. The 6E10 antibody was used to confirm whether the full-length Aβ _1-42 peptides were stably immobilized on the plate in equal amounts, and the A11 antibody was used to confirm whether the full-length Aβ _1-42 peptides aggregated during the attachment process in the well. For comparative analysis, control groups monomerized by treatment with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or sodium lauryl sulfate (SDS) for 2 hours were used.

In addition, using anti-Aβ _18-23 monoclonal 4G8 antibody and anti-Aβ _4-10 polyclonal 6E10 antibody, the structural/morphological changes of Aβ _1-42 fragments or full-length Aβ _1-42 peptides immobilized on plates were observed during the attachment process. It was confirmed whether the original traits were maintained. The epitope of 4G8 is known to be the Aβ _18-23 sequence of Aβ _1-42 , and the epitope of 6E10 is known to be the Aβ _4-10 sequence of Aβ _1-42 . Therefore, these antibodies were used to confirm whether they targeted attached Aβ _1-42 fragments or full-length Aβ _1-42 .

Additionally, 10 μM full-length Aβ _1-42 peptide was treated to confirm whether the attached full-length Aβ _1-42 maintained its unique aggregation mechanism. When thioflavin-T (ThT) fluorescent substance is inserted between the layers of the β-sheet of Aβ _1-42 aggregates, the fluorescence intensity increases and is red-biased. Using these properties of ThT, the attached full-length Aβ _1-42 peptide It was confirmed whether the additionally added 10 μM full-length Aβ _1-42 peptide coagulate with each other.

Figure 8 shows the results of confirming structural, morphological, and cohesive changes in A3 and MAP peptides.

As shown in Figure 8, when using the 6E10 antibody and A11 antibody, full-length Aβ _1-42 peptides were not found aggregated in the plate, and there was no difference when compared with the control group. It was confirmed that the 4G8 antibody targets full-length Aβ _1-42 and Aβ _18-23 . The 6E10 antibody was also confirmed to target full-length Aβ _1-42 , Aβ _4-9 , and Aβ _5-10 peptides. Through ThT fluorescence, aggregation was gradually observed during the incubation time, and the formation of amyloid fibrils with a stable β-sheet shape was confirmed after 24 hours.

Through the above results, it was confirmed that all full-length Aβ _1-42 peptides attached to the plate maintained a stable monomeric form, and that fragments and full-length Aβ _1-42 peptides maintained their unique structural/morphological characteristics even after the attachment process. confirmed.

Experimental Example 2. Screening of amyloid beta aggregation inhibitors

2.1. Screening of amyloid beta aggregation inhibitors

Fluor-Aβ _1-42 (10μM), a conjugate of the fluorescent substance Flamma 552 and Aβ _1-42, and an Aβ aggregation inhibitor were added to A3 and reacted at room temperature for 24 hours ((+)inhibitor/(+)fluor-Aβ _1-42 A3). For comparative analysis, control wells were treated with only fluor-Aβ _1-42 ((-)inhibitor/(+)fluor-Aβ _1-42 A3). Substances inhibiting Aβ aggregation were searched by comparing the fluorescence intensity of Flamma 552 between the control and wells containing the aggregation inhibitor, and these are shown in Figures 9 and 11.

To normalize the experimental data, the formula Zi = (Xi - min(x)) / (max (x) - min (x))* 100 was used.

Zi: The normalized value in the dataset

Xi: The value in the dataset, (+)inhibitor/(+)fluor-Aβ _1-42 A3

min(x): (-)inhibitor/(+)fluor-Aβ _1-42 A3

Max(x): (-)inhibitor/(-)fluor-Aβ _1-42 A3

Figure 9 is a schematic diagram showing a method for screening candidate substances as amyloid beta aggregation inhibitors.

Figure 11 is a graph confirming the aggregation inhibition effect of amyloid beta aggregation inhibitors (Curcumin and Scyllol-inositol) using A3.

As shown in Figure 11, aggregation inhibitors were searched through the Aβ aggregation inhibitor search method using A3. Curcumin was confirmed to inhibit Aβ aggregation by 49.0% and scyllo-inositol by 24.7%.

2.2. Screening of amyloid beta dissociators

Fluor-Aβ _1-42 (10 μM) was added to A3 and aggregated in the plate at 37°C for 6 hours ((-)dissociator/(+)fluor-Aβ _1-42 A3). Afterwards, Aβ aggregation dissolving agent was added and treated at room temperature for 24 hours ((+)dissociator/(+)fluor-Aβ _1-42 A3). Substances that dissolve aggregation were searched by comparing the fluorescence intensity of Flamma 552 after aggregation for 6 hours and the degree to which the fluorescence intensity of Flamma 552 was reduced after treatment with an aggregation dissolving agent, and these are shown in Figures 10 and 12.

To normalize the data, the formula Zi = (Xi - min(x)) / (max (x) - min (x))* 100 was used.

Zi: The normalized value in the dataset

Xi: The value in the dataset, (+)dissociator/(+)fluor-Aβ _1-42 A3

min(x): (-)dissociator/(+)fluor-Aβ _1-42 A3

Max(x): (-)dissociator/(-)fluor-Aβ _1-42 A3

Figure 10 is a schematic diagram showing a method for screening candidate substances as an amyloid beta aggregation dissociator.

Figure 12 is a graph confirming the aggregation-dissolving effect of amyloid beta aggregation-dissolving agents (Necrostantin-1 and Sunitinib) using A3.

As shown in Figure 12, agglutination dissolving agents were searched through the Aβ aggregation dissolving agent search method using A3. Necrostatin-1 was confirmed to dissolve Aβ aggregates by 42.5% and sunitnib by 65.2%.

Through the above results, it was confirmed that A3 and MAP can provide a platform for effectively screening Aβ aggregation inhibitors or dissolution candidates.

Experimental Example 3. Active site mapping of amyloid beta aggregation inhibitor

3.1. Active site mapping of amyloid beta aggregation inhibitor

Fluor-Aβ _1-42 (10 μM), a conjugate of the fluorescent substance Flamma 552 and Aβ _1-42, and an Aβ aggregation inhibitor were added to the MAP plate and reacted at room temperature for 24 hours ((+)inhibitor/(+)fluor -Aβ _1-42 MAP). For comparative analysis, control wells were treated with only fluor-Aβ _1-42 ((-)inhibitor/(+)fluor-Aβ _1-42 MAP). By comparing the fluorescence intensity of Flamma 552 between the control and the wells containing the aggregation inhibitor, it was confirmed which fragments the Aβ aggregation inhibitor inhibits aggregation, thereby identifying the site of action of the substance, which is shown in Figure 13.

To normalize the experimental data, the formula Zi = (Xi - min(x)) / (max (x) - min (x))* 100, the same method as in Experimental Example 2.1, was used. In addition, the degree of aggregation inhibition was analyzed for each fragment, and an average value reflecting fragments with overlapping sequences was calculated. In addition, by normalizing based on the highest value, a color-coded heatmap was created to confirm which specific sequence of Aβ the aggregation inhibitor targets (the stronger the inhibition of Aβ aggregation in green), which is shown in Figures 15 and 16. indicated.

Figure 15 is a graph confirming the aggregation-dissolving action site of the amyloid beta aggregation inhibitor (Curcumin) using MAP.

Figure 16 is a graph confirming the aggregation inhibition site of amyloid beta aggregation inhibitor (Scyllol-inositol) using MAP.

As shown in Figure 15, through the method of identifying Aβ aggregation inhibitors using MAP, curcumin was identified as HQKLVFFA (Aβ _14-19 , Aβ _15-20 , Aβ _16-21 ), GAIIGLM (Aβ _29-34 , Aβ _30-35 ), and GGVVIA (Aβ _37-42 ) domains, and was confirmed to inhibit aggregation by binding to these regions.

As shown in Figure 16, through the method of identifying Aβ aggregation inhibitors using MAP, scyllo-inositol was identified as VHHQKLVFF (Aβ _12-17 , Aβ _13-18 , Aβ _14-19 , Aβ _15-20 ), VGSNKGAIIGL (Aβ _24-29 ) , Aβ _25-30 , Aβ _26-31 , Aβ _27-32 , Aβ _28-33 , Aβ _29-34 ), LMVGGV (Aβ _34-39 ), and GGVVIA (Aβ _37-42 ) domains. , it was confirmed that aggregation was inhibited by binding to this site.

3.2. Mapping the active site of the amyloid beta dissociator.

Fluor-Aβ _1-42 (10 μM) was added to MAP and aggregated in the plate at 37°C for 6 hours ((-)dissociator/(+)fluor-Aβ _1-42 MAP). Afterwards, Aβ aggregation dissolving agent was added and treated at room temperature for 24 hours ((+)dissociator/(+)fluor-Aβ _1-42 MAP). By comparing the fluorescence intensity of Flamma 552 after aggregation for 6 hours and the degree to which the fluorescence intensity of Flamma 552 is reduced after treatment with an aggregation dissolving agent, the site of action of the substance is identified by confirming which fragments the Aβ aggregation dissolving agent dissolves. and this is shown in Figure 14.

To normalize the experimental data, the same formula as in Experimental Example 2.2, Zi = (Xi - min(x)) / (max (x) - min (x))* 100, was used. In addition, the degree of aggregation inhibition was analyzed for each fragment, and an average value reflecting fragments with overlapping sequences was calculated. In addition, by normalizing based on the highest value, a color-coded heatmap was created to confirm which specific sequence of Aβ the aggregation lytic agent targets (Aβ aggregation dissolution becomes stronger as the color increases in blue), which is shown in Figures 17 and 18. indicated.

Figure 17 is a graph confirming the aggregation-dissolving action site of amyloid beta aggregation-dissolving agent (Necrostantin-1) using MAP.

Figure 18 is a graph confirming the aggregation inhibitory action site of amyloid beta aggregation dissolving agent (Sunitinib) using MAP.

As shown in Figure 17, through the method of identifying Aβ aggregation dissolving agents using MAP, necrostatin-1 was identified as HHQKLVFF (Aβ _13-18 , Aβ _14-19 , Aβ _15-20 ) and GLMVGGVVIA (Aβ _33-38 , Aβ _34-39 ). , Aβ _35-40 , Aβ _36-41 , and Aβ _37-42 ) domains and can be confirmed to cause aggregation and dissolution. It was confirmed that the Aβ _36-41 region was specifically released when compared to the full-length Aβ _1-42 .

As shown in Figure 18, through the method of identifying Aβ aggregation inhibitors using MAP, sunitnib was identified as AIIGLM (Aβ _30-35 ), and LMVGGVVIA (Aβ _34-39 , Aβ _35-40 , Aβ _36-41 , Aβ _37-42 ). It was confirmed that it specifically targets domains and inhibits aggregation.

Based on the above results, MAP can be useful in analyzing the target domains of Aβ aggregation inhibitors or solubilizers and developing inhibitors or solubilizers that unravel the aggregation of specific protein sequences of Aβ.

Claims (24)

1. (1) providing a plate to which the first amyloid protein is attached;

   (2-1) contacting the plate with a second amyloid protein bound to a protein aggregation inhibitor candidate and an indicator; or

   (2-2) contacting the second amyloid protein bound with an indicator substance to the plate and then treating the candidate substance with a protein aggregation dissolving agent; and,

   (3-1) measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-1) and selecting aggregation inhibitor candidates changed compared to the untreated control group; or

   (3-2) A protein aggregation inhibitor or aggregation-dissolving agent comprising the step of measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-2) and selecting candidates for aggregation-dissolving agent changed compared to the untreated control group. How to screen.
2. The method according to claim 1, wherein step (2-1) or (2-2) further comprises the step of inducing aggregation of the first and second amyloid proteins.
3. The method according to claim 1, wherein the amyloid protein is amyloid beta, tau protein, or alpha-synuclein (α-cynuclein).
4. The method according to claim 1, wherein the first and second amyloid proteins self-assemble.
5. The method according to claim 1, wherein the first or second amyloid protein is a full-length protein or a protein fragment.
6. The method of claim 5, wherein the protein fragment is a contiguous 6 to 15 mer within an amyloid protein.
7. The method of claim 1, wherein the first amyloid protein is attached to the plate through cysteine.
8. The method of claim 1, wherein the first amyloid protein is attached to the plate to maintain its monomeric form.
9. The method according to claim 1, wherein the indicator substance is a fluorescent substance.
10. The method of claim 1, wherein the aggregation inhibitor candidate is treated simultaneously or sequentially with the second amyloid protein.
11. The method according to claim 1, wherein the aggregation-dissolving agent candidate is treated after the first amyloid protein and the second amyloid protein are aggregated.
12. The method according to claim 1, wherein the first amyloid protein of (1) is treated on the plate at a concentration in the range of 0.1 μM to 10 μM or for a time in the range of 20 hours to 26 hours.
13. The method according to claim 1, wherein the first amyloid protein of (1) is treated on a plate at a temperature in the range of 25°C to 35°C or pH conditions in the range of pH 5 to pH 8.
14. The method according to claim 1, wherein the second amyloid protein of (2-1) or (2-2) is prepared at a concentration in the range of 5 μM to 15 μM, a temperature in the range of 28 ℃ to 38 ℃, or pH conditions in the range of pH 5 to pH 8. 1 A method of contacting a plate to which amyloid protein is attached.
15. The method of claim 1, wherein the aggregation inhibitor or aggregation dissolving agent inhibits or dissolves aggregation of amyloid beta, tau protein, or alpha-synuclein.
16. The method according to claim 1, wherein the aggregation inhibitor or aggregation-dissolving agent is a treatment for degenerative brain disease.
17. The method of claim 14, wherein the degenerative brain disease includes dementia, Alzheimer's disease, preclinical Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, Cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinal cord Spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia , progressive supranuclear palsy, and frontotemporal dementia.
18. A plate to which the first amyloid protein is attached; and

    Contains a second amyloid protein to which an indicator substance is bound,

    A kit for screening a protein aggregation inhibitor or aggregation dissolving agent, wherein the first and second amyloid proteins are self-assembled.
19. The kit according to claim 18, wherein the first amyloid protein is a full-length protein or a protein fragment.
20. (1) providing a plate to which a plurality of first amyloid protein fragments are attached;

    (2-1) contacting the plate with a second amyloid protein bound to a protein aggregation inhibitor candidate and an indicator; or

    (2-2) contacting the second amyloid protein bound with an indicator substance to the plate and then treating the candidate substance with a protein aggregation dissolving agent;

    (3-1) measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-1); or

    (3-2) Screening the site where the amyloid protein aggregation inhibitor candidate or aggregation dissolver candidate acts, including the step of measuring the aggregation rate of the first amyloid protein and the second amyloid in step (2-2). method.
21. The method of claim 20, wherein step (2-1) or (2-2) further comprises inducing aggregation of the first and second amyloid proteins.
22. The method of claim 20, wherein the amyloid protein is amyloid beta, tau protein, or alpha-synuclein.
23. The method of claim 20, wherein the protein fragment is a contiguous 6 to 15 mer in an amyloid protein, and the plurality of protein fragments include two or more consecutive overlapping amino acid residues.
24. The method of claim 23, further comprising determining a protein fragment for which a high degree of aggregation inhibition or aggregation dissolution is measured as the site of action of the candidate substance.

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