Visualization agent for visualizing aggregation state of protein and method for using visualization agent

Abstract

A visualization agent for visualizing the aggregation state of a protein according to an embodiment contains a chemical probe molecule that binds to the protein in an aggregated state to form a complex. The chemical probe molecule is an aminocoumarin analog having a leaving group that leaves due to the formation of the complex. A method for visualizing the aggregation state of a protein according to an embodiment comprises: a contact step for bringing a visualization agent into contact with an aggregated protein; and a detection step for detecting the fluorescence of a complex of the aggregated protein and the visualization agent.

Description

Visualizing agent for visualizing the aggregation state of proteins and method for using the same

[0001] This invention relates to a visualization agent for the aggregation state of proteins and a method for using the same.

[0002] Proteins are known to change into aggregated states, such as amyloid fibrils, under heating or specific conditions. Protein aggregation is an important phenomenon in basic protein research and is deeply involved in neurodegenerative diseases. Therefore, the development of visualization agents and methods for using them to visualize protein aggregation will have a significant academic and industrial impact.

[0003] As a conventional visualization agent for the aggregation state of proteins, a nitrobenzoxadiazole (NBD) type visualization agent, represented by the following formula, has been disclosed (Patent Document 1).

[0004] International Publication No. 2023 / 140228

[0005] By concentrating aggregated proteins labeled with a visualization agent, highly sensitive measurements become possible. For NBD-type visualization agents, a method using anti-NBD antibodies to concentrate labeled aggregated proteins is employed. However, the concentration efficiency is insufficient, and only a portion of the labeled aggregated proteins can be recovered. As a result, high-intensity signals cannot be obtained during measurement, leading to insufficient measurement sensitivity.

[0006] The objective is to provide a visualization agent and a method for using it for detecting and visualizing aggregated proteins with high sensitivity.

[0007] The visualization agent according to the present invention, which solves the above problems, includes a chemical probe molecule that binds to aggregated proteins and forms a complex. Furthermore, the chemical probe molecule is an aminocoumarin analog having a leaving group that is released upon the formation of the complex.

[0008] Furthermore, a method for using the visualization agent according to the present invention to solve the above problems includes the steps of: contacting an aggregated protein with a visualization agent that binds to the aggregated protein and forms a complex to obtain a mixture; and detecting the complex in the mixture.

[0009] Furthermore, as a method using the visualization agent according to the present invention, a method for identifying aggregated proteins that bind to a test compound is provided. The method comprises the following steps: (1) a step of preparing a sample containing multiple types of proteins, a test compound, and a visualization agent that binds to aggregated proteins to form a complex; (A1) a step of bringing the sample, the test compound, and the visualization agent prepared in step (1) into contact with each other to obtain a first mixture; (A2) a step of performing a protein denaturation operation on the first mixture after step (A1); (A3) a step of isolating and purifying each protein in the first mixture after step (A2); (A4) a step of subjecting each protein isolated and purified in step (A3) to mass spectrometry; (B1) a step of bringing the sample and the visualization agent prepared in step (1) into contact with each other to obtain a second mixture; (B2) a step of performing a protein denaturation operation on the second mixture obtained in step (B1); (B3) a step of isolating and purifying each protein contained in the second mixture obtained in step (B2). (B4) A step of subjecting each of the proteins isolated and purified in step (B3) to mass spectrometry; (10) A step of comparing the measurement results of step (A4) and step (B4), and identifying proteins whose signal intensity measured in step (A4) is lower than the signal intensity measured in step (B4) as aggregated proteins that bind to the test compound.

[0010] The visualization agent and method of the present invention can be used in protein-mixed systems and applied to proteomics analysis because the fluorescent chemical probe molecule covalently binds to aggregated proteins and remains as a record of aggregation. Furthermore, it can visualize the aggregation state of proteins with higher sensitivity than conventional fluorescent chemical probe molecules.

[0011] Figure 1 is a graph showing the change in fluorescence intensity of the product obtained by reacting the chemical probe molecule of the visualization agent of the first embodiment with Ethylamine (solid line) and the change in fluorescence intensity of the chemical probe molecule before the reaction (dashed line). Figure 2 is a graph showing the signal intensity derived from the detected ion obtained by subjecting the visualization agent of the first embodiment to mass spectrometry. Figure 3 is a diagram showing the change due to the progression of amyloid-β protein aggregation. Figure 4 is a flowchart showing the method of the second embodiment. Figure 5 is a flowchart showing the method of the third embodiment. Figure 6 is a flowchart showing the method of the fourth embodiment. Figure 7 is a flowchart showing the method of the fifth embodiment. Figure 8 is a diagram showing the experimental results of Example 1. Figure 9 is a diagram showing the experimental results of Example 1. Figure 10 is a diagram illustrating the experimental design of Example 2. Figure 11 is a diagram showing the experimental results of Example 2. Figure 12 is a diagram showing the experimental results of Example 2. Figure 13 is a diagram showing the experimental results of Example 2. Figure 14 is a diagram showing the experimental results of Example 2. Figure 15 is a diagram showing the experimental results of Example 2. Figure 16 shows the experimental results for Example 2. Figure 17 is a diagram illustrating the experimental design for Example 3. Figure 18 shows the experimental results for Example 3. Figure 19 shows the experimental results for Example 4. Figure 20 shows the experimental results for Example 5. Figure 21 shows the experimental results for Example 5. Figure 22 shows the experimental results for Example 6. Figure 23 shows the experimental results for Example 7. Figure 24 shows the experimental results for Example 7. Figure 25 shows the experimental results for Example 7. Figure 26 shows the experimental results for Example 8. Figure 27 shows the experimental results for Example 9.

[0012] Embodiments of the present invention will be described in detail below with reference to the drawings. Each figure is a schematic diagram illustrating the embodiment and its purpose to facilitate understanding. While the shape, dimensions, ratios, etc., may differ from those of the actual product, these can be appropriately modified in accordance with the following description and known technology.

[0013] In this specification, "linear alkyl group having 1 to 12 carbon atoms" refers to, for example, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, an n-decyl group, an n-undecyl group, and an n-dodecyl group.

[0014] In this specification, "branched hydrocarbon group having 3 to 10 carbon atoms" refers to, for example, iso-propyl group, iso-butyl group, sec-butyl group, tert-butyl group, iso-pentyl group, sec-pentyl group, tert-pentyl group, 3-pentyl group, neopentyl group, iso-hexyl group, sec-hexyl group, tert-hexyl group, neohexyl group, iso-heptyl group, sec-heptyl group, tert-heptyl group, neoheptyl group, iso-octyl group, sec-octyl group, tert-octyl group, neooctyl group, iso-nonyl group, sec-nonyl group, tert-nonyl group, neonyl group, iso-decyl group, sec-decyl group, tert-decyl group, or neodecyl group.

[0015] In this specification, "alicyclic hydrocarbon group having 3 to 10 carbon atoms" refers to, for example, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, or a cyclodecyl group.

[0016] In this specification, "aryl group having 3 to 10 carbon atoms" refers to, for example, a benzyl group, a phenyl group, a 1-naphthyl group, or a 2-naphthyl group.

[0017] [First Embodiment] (Visualizing Agent) The visualizing agent of the first embodiment includes a chemical probe molecule that binds to aggregated proteins and forms a complex. The chemical probe molecule is an aminocoumarin analog having a leaving group that is released upon the formation of the complex. The visualizing agent may be either a compound or a composition, as long as it visualizes the aggregated state of proteins with high sensitivity. That is, the visualizing agent may be the chemical probe molecule itself, or it may also include compounds other than the chemical probe molecule (e.g., pH adjusters, stabilizers, preservatives, etc.).

[0018] In this specification, "visualization of protein aggregation state" means making the aggregation state of a protein discernible under various conditions and methods (not limited to visual inspection). For example, determining whether a target protein is an aggregated protein or determining the degree to which a target protein is aggregated (i.e., quantitative determination of the degree of aggregation) also falls under "visualization of protein aggregation state."

[0019] In this specification, "aminocoumarin analogue" refers to a compound having a coumarin skeleton that contains an amino group as a functional group. In "aminocoumarin analogues," the amino group may be substituted at any of the 3rd to 8th positions of the coumarin skeleton.

[0020] The aminocoumarin analog, which is a chemical probe molecule contained in the visualization agent of this embodiment, has a leaving group as a functional group other than the amino group. This leaving group is a functional group (other than the amino group) of the coumarin skeleton that is released when the aminocoumarin analog, which is the chemical probe molecule, forms a complex with the aggregated protein. The chemical probe molecule may also be an aminocoumarin analog having a coumarin skeleton with functional groups other than the amino group and the leaving group.

[0021] Because chemical probe molecules have a coumarin backbone, the overall physical properties of the probe are hydrophobic. On the other hand, the surface of non-denatured or non-aggregated proteins is predominantly composed of hydrophilic amino acids, so the amino acid residues exposed on the protein surface are generally hydrophilic. Therefore, chemical probe molecules hardly bind to non-denatured or non-aggregated proteins. However, when a protein is denatured or aggregated, the proportion of hydrophobic regions where hydrophobic amino acids are exposed on the protein surface (e.g., at the aggregation interface) increases, and the protein surface as a whole becomes hydrophobic. Chemical probe molecules exert hydrophobic interactions with the hydrophobic regions of aggregated proteins, resulting in a longer residence time near the hydrophobic regions of aggregated proteins than when they are near non-denatured or non-aggregated proteins. Aggregated proteins may have highly hydrophilic lysine residues around their hydrophobic regions (e.g., one or two residues away), but because of the longer residence time, covalent bonds are easily formed between the chemical probe molecule and the lysine residues due to the proximity effect. In other words, chemical probe molecules have a higher binding affinity to aggregated proteins compared to non-denatured or non-aggregated proteins, and more selective and specific binding is easily formed.

[0022] Chemical probe molecules bind to lysine residues of aggregated proteins to form complexes. However, lysine residues near neutral pH are thought to exist in a large proportion of the less nucleophilic cationic form. Chemical probe molecules do not normally react readily with such cationic lysine residues. However, if they reside near the lysine residue for a long time and are in close proximity, a specific structure in the coumarin skeleton of the chemical probe molecule may function as a base, potentially increasing the nucleophilicity of the lysine residue and making it easier for the aforementioned leaving group to detach.

[0023] The leaving ability (i.e., reactivity) of the leaving group is preferably moderate from the viewpoint of selectivity for aggregated proteins. If the reactivity of the leaving group is too low, the efficiency of labeling decreases, making it difficult to measure with high sensitivity. On the other hand, if the reactivity is too high, accidental binding to lysine residues increases, reducing selectivity for aggregated proteins.

[0024] The chemical probe molecule and its reactant (in this case, a complex with aggregated protein) have a coumarin skeleton and can therefore emit fluorescence. In other words, the chemical probe molecule is a fluorescent substance and continues to emit fluorescence even after binding to the aggregated protein, so it can also be used for fluorescent labeling of aggregated proteins. Therefore, the visualization agent of the first embodiment can also be used for selective visualization of aggregated proteins.

[0025] Furthermore, the fluorescence intensity of the chemical probe molecule is greatly enhanced by its reaction with aggregated protein. Figure 1 shows the change in fluorescence intensity between the product obtained by reacting the chemical probe molecule with ethylamine, which is a mimic of the reaction product of the chemical probe molecule with a Lys residue, and the chemical probe molecule before the reaction. In other words, whether or not aggregated protein has bound to the chemical probe molecule of the visualization agent, and the degree of binding affinity, can be detected by measuring the fluorescence intensity at the excitation wavelength (=365 nm) (fluorescence wavelength = approximately 450 nm).

[0026] The proteins to be visualized, i.e., the samples, are not particularly limited, and any protein that may be aggregated can be used for visualization. Since aggregation can occur due to changes in protein structure caused by thermal denaturation, heated proteins may also be used for visualization. Furthermore, proteins that may have undergone denaturation due to causes other than heat, such as denaturation by denaturing agents, acid denaturation, and pressure denaturation, may also be used for visualization. Specific examples of proteins that may aggregate include amyloid-beta protein (Aβ40 or Aβ42), tau, α-synuclidein, carbonic anesthetic (CA) II, and trastuzumab.

[0027] The method for contacting the aggregated protein with the chemical probe molecule is not particularly limited; for example, it could involve adding the chemical probe molecule to any suspension containing the aggregated protein.

[0028] The amount of chemical probe molecules used is not particularly limited, but is usually 10 to 10,000 mol per mol of protein to be visualized, and preferably 10 to 100 mol.

[0029] The chemical probe molecule preferably has a coumarin skeleton containing a tertiary amino group as a functional group among aminocoumarin analogs. This is because the ionization tendency of the chemical probe molecule is higher than when it contains a primary amino group or a secondary amino group as a functional group, and it is easier to obtain a high-intensity signal in various detection methods of the chemical probe molecule and can be measured with high sensitivity. The high-intensity signal in detection means, for example, a larger signal intensity in mass spectrometry. For example, FIG. 2 shows the ion intensity observed in mass spectrometry accompanied by ionization, and it can be seen that CAP-C-PF shows a higher ion intensity than Octyl-O-NBD.

[0030] When the chemical probe molecule is an aminocoumarin analog having a coumarin skeleton containing a tertiary amino group as a functional group, the visualizing agent may contain a compound represented by the following general formula (I) as the chemical probe molecule.

[0031] In the formula, R and R' are each independently selected from the group consisting of a chain hydrocarbon group having 1 to 12 carbon atoms, a branched hydrocarbon group having 3 to 10 carbon atoms, an alicyclic hydrocarbon group having 3 to 10 carbon atoms, and an aryl group having 3 to 10 carbon atoms (however, one or more -CH ２ -CH ２ - in the chain hydrocarbon group is substituted with -CO-NH- or -CO-O-, or one or more -CH ２ -CH ２ -CH ２ - in the chain hydrocarbon group is substituted with -NH-CO-NH- or -O-CO-NH-, or one carbon atom or two or more non-adjacent carbon atoms in the branched hydrocarbon group or the alicyclic hydrocarbon group may be substituted with a nitrogen atom or an oxygen atom), and X is a phenyl group in which the ortho position is substituted with a fluorine atom (for example, a trifluorophenoxy group, a tetrafluorophenoxy group or a pentafluorophenoxy group, etc.), a benzoxazole ring, a benzothiazole ring, or a pyrimidine ring.

[0032] Hereinafter, each atomic group constituting the chemical formula of the above formula (I) will be described in detail.

[0033] R and R' are each independently selected atomic groups from the groups described in the above general formula. For example, when R is a chain hydrocarbon group, R' may be the same chain hydrocarbon group as R, a different chain hydrocarbon group, or a branched hydrocarbon group, an alicyclic hydrocarbon group, or an aryl group.

[0034] When R and / or R' is a chain hydrocarbon group, the chain hydrocarbon group is, for example, a saturated hydrocarbon group, that is, a linear alkyl group. Alternatively, R and / or R' may be a chain hydrocarbon group and an unsaturated hydrocarbon group.

[0035] When R and / or R' is a chain hydrocarbon group, the number of carbon atoms of the chain hydrocarbon group may be any of 1 to 12, but preferably, it is selected from 2 to 8 carbon atoms. More preferably, R and / or R' is an n-butyl group or an n-octyl group, and even more preferably an n-octyl group.

[0036] Among them, when R and / or R' is a chain hydrocarbon group containing -CH ２ -CH ２ -(that is, having 2 or more carbon atoms, preferably 3 or more carbon atoms), the -CH ２ -CH ２ - may be substituted with -CO-NH- or -CO-O-. This substitution is included one or more times in R and / or R' (for example, it may be only 1, 2, or 3), and may be substituted continuously.

[0037] Among them, when R and / or R' is a chain hydrocarbon group containing -CH ２ -CH ２ -CH ２ -(that is, having 3 or more carbon atoms, preferably 4 or more carbon atoms), -CH ２ -CH ２ -CH ２ - may be substituted with -NH-CO-NH- or -O-CO-NH-. This substitution is included one or more times in R and / or R' (for example, it may be only 1, 2, or 3), and may be substituted continuously.

[0038] If R and / or R' are branched hydrocarbon groups, the chain hydrocarbon group is, for example, a saturated hydrocarbon group, i.e., a linear alkyl group. Alternatively, R and / or R' may be both a chain hydrocarbon group and an unsaturated hydrocarbon group.

[0039] When R and / or R' are branched hydrocarbon groups, the number of carbon atoms in the branched hydrocarbon group can be any of 3 to 10, but preferably selected from 3 to 8. More preferably is an iso-propyl group, a sec-butyl group, a tert-butyl group, or a 3-pentyl group, and even more preferably iso-propyl group. One carbon atom or two or more non-adjacent carbon atoms in the branched hydrocarbon group may be substituted with a nitrogen atom or an oxygen atom. This substitution may be one, two, or three in the branched hydrocarbon group. A specific example of a branched hydrocarbon group in which carbon atoms are substituted with nitrogen atoms or oxygen atoms is the 1-methyl-2-methylaminoethyl group.

[0040] If R and / or R' are alicyclic hydrocarbon groups, the number of carbon atoms in the alicyclic hydrocarbon group may be any number from 3 to 10, but preferably selected from 3 to 8 carbon atoms. More preferably, it is a cyclohexyl group. One carbon atom or two or more non-adjacent carbon atoms in the alicyclic hydrocarbon group may be substituted with a nitrogen atom or an oxygen atom. This substitution may be one, two, or three in the alicyclic hydrocarbon group. Specific examples of alicyclic hydrocarbon groups in which carbon atoms are substituted with nitrogen atoms or oxygen atoms include 3-azetidyl group, 3-tetrahydrofuranyl group, 3-piperidinyl group, and 4-tetrahydropyranyl group. The alicyclic hydrocarbon group may be substituted with substituents. Examples of substituents include amino groups and aminomethyl groups. Specific examples of alicyclic hydrocarbon groups substituted with substituents include 3-aminomethylcyclobutyl group, 3-aminocyclopentyl group, and 4-aminocyclohexyl group.

[0041] If R and / or R' are aryl groups, the number of carbon atoms in the aryl group can be any number from 3 to 10, but preferably selected from 5 to 7. More preferably, it is a benzyl group or a phenyl group. The aryl group may also be substituted with a substituent. Examples of substituents include amino groups and aminomethyl groups.

[0042] As mentioned above, X is a phenyl group with a nitrogen atom added to the ortho position (e.g., a trifluorophenoxy group, a tetrafluorophenoxy group, or a pentafluorophenoxy group), a benzoxazole ring, a benzothiazole ring, or a pyrimidine ring. This atomic group is eliminated during the process in which the chemical probe molecule forms a complex with the aggregated protein. In other words, X functions as a leaving group in binding to the aggregated protein. The nucleophilic substitution reaction with the lysine residue of the aggregated protein is assumed to be specifically the reaction mechanism shown in formula (II) below.

[0043]

[0044] The leaving property of X is strongly influenced by its electrophilicity. The degree of electrophilicity can be converted using the acid dissociation constant (pKa) of its conjugate acid. For X to exhibit appropriate leaving property, its pKa is preferably 6.0 or less. More preferably, the pKa of the conjugate acid of X is 5.5 or higher and 5.7 or lower. By using the compound of formula (I) above, having such an X, as a chemical probe molecule, the formation of a complex between the visualization agent and the aggregated protein becomes more selective.

[0045] R' may further have a site to which a click reaction can be applied (hereinafter referred to as the "click reaction site"). The click reaction site is, for example, a carbon-carbon triple bond, a cyclooctin structure or a cyclooctene structure, or an azide group or a tetrazine structure. In other words, the click reaction site is selected from the group consisting of a carbon-carbon triple bond, a cyclooctin structure and a cyclooctene structure, or from the group consisting of an azide group and a tetrazine structure.

[0046] (Diagnostic Agent) The visualization agent of the first embodiment can also be used as a diagnostic agent for Alzheimer's disease. This will be explained below.

[0047] Amyloid-beta protein undergoes a transition from oligomers in the initial stages of aggregation to protofibrils and ultimately to fibrils as aggregation progresses (see Figure 3). Amyloid-beta protein is soluble from the oligomer to the protofibril stage, but becomes insoluble once it transforms into fibrils. The antibody drug recanemab, which targets aggregated amyloid-beta protein, targets protofibrils, i.e., soluble aggregates. Furthermore, it has been reported that soluble aggregates are more toxic to nerve cells than insoluble aggregates such as fibrils. For these reasons, there has been a need for a technology that can observe soluble aggregates of amyloid-beta protein.

[0048] Thioflavin T (ThT), a conventional research reagent for amyloid-beta protein, is known to stain amyloid-beta protein fibrils but does not respond to the oligomeric state. Furthermore, NBD-type visualizers, which are conventional visualization agents for aggregated proteins, react with the oligomeric state, the initial aggregated state of amyloid-beta protein, because their operating principle differs from that of thioflavin T. This indicates that amyloid-beta protein is more reactive with NBD-type visualizers and can be labeled in the soluble oligomeric state than in the fibril state. On the other hand, as mentioned above, NBD-type visualizers have the drawback of insufficient enrichment efficiency of labeled aggregated proteins.

[0049] Furthermore, Tau441(2N4R)P301S is known as a biomarker associated with Alzheimer's disease. Conventional visualization agents for Tau441(2N4R)P301S also had the same problems as conventional visualization agents for amyloid-beta protein.

[0050] The inventors have revealed that the visualization agent of the first embodiment (particularly CAP-C-PF-alkyne) preferentially labels the initial aggregation state of amyloid-beta protein. Furthermore, the inventors have also revealed that the visualization agent of the first embodiment (particularly CAP-C-PF-alkyne) preferentially labels fibril-like Tau441(2N4R)P301S (as will be explained in the experimental examples below). In other words, the visualization agent of the first embodiment can be used as a diagnostic agent for Alzheimer's disease.

[0051] Furthermore, the inventors have also revealed that the visualization agent of the first embodiment (particularly CAP-C-PF-alkyne) preferentially labels fibril-like α-synuclein (see experimental examples described later). In other words, the visualization agent of the first embodiment (particularly CAP-C-PF-alkyne) can also be used as a diagnostic agent for Parkinson's disease.

[0052] The visualization agent of the first embodiment may include a click reaction site, as described above. When the visualization agent has a click reaction site, it can be concentrated with high efficiency, for example, by biotinylation and recovery with avidin beads. Therefore, the visualization agent of the first embodiment enables more accurate diagnosis in applications as a diagnostic agent for Alzheimer's disease or Parkinson's disease.

[0053] The diagnostic agent of the first embodiment is applied to a sample derived from the subject (the "subject" will be described in detail in the second embodiment). The diagnostic agent is used, for example, in vitro to contact a sample derived from the subject and specifically label amyloid-beta protein or Tau441(2N4R)P301S. By determining the presence or absence of the labeled protein using the diagnostic agent, it is possible to diagnose whether or not the subject has Alzheimer's disease. If the diagnostic agent has a click reaction site, the labeled protein may be reacted with a biotinylation reagent and concentrated with avidin beads. After such a concentration operation, the presence or absence of the labeled protein may be determined by mass spectrometry.

[0054] [Second Embodiment] (Method for Visualizing Agglutinated Proteins) The second embodiment relates to a method for visualizing aggregated proteins in a sample, as shown in Figure 4. Specifically, the method for visualizing aggregated proteins in a sample includes the steps of: contacting the sample with the visualization agent of the first embodiment (contact step (S21)); and detecting a complex of the visualization agent and aggregated proteins in the sample (detection step (S22)).

[0055] While a sample containing only one type of protein may be used, typically a sample containing a mixture of multiple proteins is used. The sample may be in solid or liquid form. That is, the protein may be contained in a liquid sample, such as body fluids, or in a solid sample, such as cells or tissues.

[0056] In the contact step (S21), the method of bringing the sample into contact with the visualization agent is not particularly limited. For example, this could involve adding a liquid containing the visualization agent to the sample, or adding a solid visualization agent to the sample.

[0057] The detection step (S22) may be fluorescence measurement. Specifically, step (S22) may be a step that detects an increase in fluorescence intensity at a specific wavelength, and can be performed according to a conventional method.

[0058] [Third Embodiment] (Method for purifying proteins that bind to a test compound) The third embodiment relates to a method for purifying proteins that bind to a test compound, as shown in Figure 5. Specifically, it includes the steps of: preparing a visualization agent that selectively binds to aggregated proteins to form a complex and a sample containing multiple types of proteins (S30); contacting the sample with the visualization agent to obtain a mixture (S31); isolating and purifying the proteins in the mixture according to their type (S32); and detecting the complex from the isolated and purified proteins (S33).

[0059] The method for contacting the sample with the visualization agent in step (S31) is not particularly limited, similar to step (S21) of the method in the second embodiment. For example, this could involve adding a liquid containing the visualization agent to the sample, or adding a solid visualization agent to the sample.

[0060] Step (S32) can be carried out by methods commonly used for the isolation and purification of proteins, such as electrophoresis or gel filtration. A method suitable for the purpose of identifying the protein, the required degree of purification, and the protein detection method should be adopted.

[0061] First, the principle of the purification method of the third embodiment will be explained. Many compounds that bind to proteins are known; for example, thioflavin T is known to bind to amyloid-beta, an aggregated protein. Most of these known compounds bind to proteins non-covalently, so their binding to proteins is easily released when separation steps such as electrophoresis are performed. Therefore, even if it is possible to determine whether aggregated proteins are present in a sample using known compounds, it is not possible to identify which protein is the aggregated protein if the sample contains multiple proteins.

[0062] On the other hand, as can be seen from the assumption that the visualization agent of the first embodiment forms a covalent bond with the lysine residue of the aggregated protein, it has been confirmed that it does not easily detach from the aggregated protein. Therefore, even if the sample contains multiple proteins, the proteins can be isolated and purified using various separation operations, including electrophoresis, and the properties of each protein can be verified. In particular, in step (S33), by detecting the complex of the visualization agent and the aggregated protein for each separated protein, it is possible to identify which protein is the aggregated protein.

[0063] Step (S33) may be carried out by detecting the fluorescence wavelength of each protein obtained in step (S32). This fluorescence detection can be performed according to a conventional method.

[0064] In a further embodiment, if the chemical probe molecule of the visualization agent is a compound of formula (I) and R' has a click reaction site, a click reaction step may be included after step (S31) and before step (S32). The click reaction step is a step in which a click reaction is carried out by adding a click reaction reagent to the mixture obtained in step (S31). Step (S32) is performed to isolate and purify by reacting with the functional group introduced in the click reaction step.

[0065] A click reaction reagent is a compound that forms a covalent bond via a click reaction between the chemical probe molecule and the click reaction site in the complex of the aggregated protein. As mentioned above, the click reaction site is selected from the group consisting of carbon-carbon triple bonds, cyclooctin structures, and cyclooctene structures, or from the group consisting of azide groups and tetrazine structures. However, the click reaction reagent contains atomic groups selected from the group not selected as click reaction sites. Furthermore, since the click reaction reagent can constitute a desired functional group, the desired functional group can be introduced into the complex of the chemical probe molecule and aggregated protein by going through the click reaction process.

[0066] The click reaction reagent may contain a "concentration tag molecule" as a desired functional group. A concentration tag molecule is a molecule that is recognized and acts as a linker or scaffold in operations that increase the concentration of a complex of chemical probe molecules and aggregated proteins in a suspension, such as biotin.

[0067] By labeling with biotin using a click reaction reagent, a complex of chemical probe molecules and aggregated proteins can be adsorbed onto avidin, which exhibits extremely high affinity for biotin. The biotin-avidin interaction is known to be very strong and rapidly formed among known non-covalent interactions between proteins and ligands, and is therefore hardly affected by extreme pH values, temperatures, polar organic solvents, or denaturants. Accordingly, in further embodiments, it is preferable to perform a concentration operation on the complex in step (S32).

[0068] If step (S32) includes the concentration operation, high-sensitivity measurement is possible. In particular, it is preferable because although a sample of a predetermined concentration is required, mass spectrometry that allows for high-sensitivity measurement can be performed. That is, step (S33) in further embodiments may be carried out by mass spectrometry. Furthermore, since the sample preparation method for proteomics analysis of biotin-labeled proteins and peptide fragments is already an established technology, high-quality proteomics analysis data can be obtained.

[0069] [Fourth Embodiment] (Method for Diagnosing Alzheimer's Disease) The fourth embodiment relates to a method for diagnosing Alzheimer's disease. Each step of this diagnostic method can be performed with the same operations as the corresponding steps in the method of the third embodiment. However, the method of the fourth embodiment differs from the method of the third embodiment in that two types of samples are prepared. The two types of samples are a sample derived from the subject and a sample derived from someone other than the subject (hereinafter referred to as "control"). Furthermore, the diagnostic method of the fourth embodiment compares the measurement results of these two types of samples to determine whether or not the subject has Alzheimer's type dementia.

[0070] Specifically, the diagnostic method of the fourth embodiment is as shown in Figure 6: (S40) A step of preparing a diagnostic agent that selectively binds to aggregated proteins to form a complex, a sample derived from the subject containing multiple types of proteins, and a sample derived from a control containing multiple types of proteins; (S41) A step of contacting the sample derived from the subject with the visualization agent prepared in step (S40) to obtain a target mixture; (S42) A step of isolating and purifying each protein in the target mixture; (S43) A step of detecting the complex from each protein isolated and purified in step (S42); (S41a) A step of contacting the sample derived from the control with the visualization agent prepared in step (S40) to obtain a control mixture; (S42a) A step of isolating and purifying each protein in the control mixture; (S43a) A step of detecting the complex from each protein isolated and purified in step (S42a); and (S44) The process includes comparing the detection results obtained in steps (S43) and (S43a) to determine whether or not the subject has Alzheimer's disease.

[0071] The diagnostic agent prepared in step (S40) is the same as the diagnostic agent described as a further embodiment of the first embodiment. That is, the diagnostic agent prepared in step (S40) is the same as the visualization agent described in the first embodiment, and consequently, the visualization agent prepared in step (S30) of the third embodiment.

[0072] Herein, “subject of test” as used herein refers to, for example, a test animal or any animal cells or tissues thereof. “Test animal” refers to mammals such as humans, monkeys, cattle, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, and rats, as well as birds such as chickens, and is preferably a mammal. “Animal cells or tissues thereof” refers to, for example, cells derived from a test animal or body tissue derived from a test animal, and is preferably cells or tissues derived from a mammal. Cells derived from a test animal refer to, for example, cells isolated from the test animal, pluripotent stem cells produced using cells isolated from the test animal, and cells differentiated from said stem cells.

[0073] "Sample" refers to any form of sample that may contain amyloid-beta protein in its initial aggregated state and / or fibrilized Tau441(2N4R)P301S, but is an in vivo sample. Examples of in vivo samples include samples taken from test animals, such as cell culture supernatants or body fluid samples. In one example, the sample used in the method of this embodiment is a human-derived sample.

[0074] "Cell culture supernatant" refers to the supernatant of a culture obtained by culturing cells of any state derived from a test subject in a culture medium for a certain period of time. Examples of cells that can be used to prepare the culture supernatant include cells isolated from the body of a test animal, cultured cells thereof, or pluripotent stem cells derived from cells isolated from the body of a test animal, or cells differentiated from said pluripotent stem cells. The cell culture conditions are, for example, 100 ml of cell culture medium in a culture dish. ５ ~10 ７ This is the culture supernatant of a culture obtained by seeding individual cells and culturing them for more than one day.

[0075] "Body fluid" refers to a liquid biological sample that can be collected from the body of an animal being tested. Examples of body fluids include blood, plasma, serum, bile, saliva, urine, tears, sweat, and cerebrospinal fluid (CSF). Preferably, the body fluid is urine or serum.

[0076] The subjects of the test may be individuals whose presence or absence of Alzheimer's disease is known, or individuals whose presence or absence of the disease is unknown. More specifically, the subjects of the test in the method of the fourth embodiment may be patients suspected of having Alzheimer's disease, or they may be derived from patients receiving treatment for Alzheimer's disease.

[0077] In this embodiment, "control" refers to a test animal that is the same individual as the test subject or a different individual, or any animal cells or tissue derived from the test animal. When the control is a different individual from the test subject, the "control" may be an individual of the same species or an individual of a different species. Here, an individual of the same species refers to an individual belonging to the same species phylogenetically. On the other hand, an individual of a different species refers to an individual belonging to a different species from the test subject. However, it is preferable that the phylogenetic classification of the individual of a different species does not differ significantly from that of the test subject, for example, it is preferable that it be of the same genus as the test subject.

[0078] When the control is the same individual as the subject, the "control-derived sample" is, for example, a sample collected under different conditions than the sample derived from the subject. Different conditions include, for example, a different organ from which the sample is collected and a different timing of collection. In other words, the control may be the subject at a past point in time. For example, if a different condition is that an individual is given some treatment that may result in a reduction of amyloid-beta protein, the individual at the time after the treatment may be used as the subject, and the individual at the time before the treatment may be used as the control.

[0079] Furthermore, if the control is a different individual, the control may be an individual whose presence or absence of Alzheimer's disease is known. Alternatively, the control may be the same individual as the subject, and may be the subject at the time when they were previously diagnosed with Alzheimer's disease. Or, the control may be the same individual as the subject diagnosed with Alzheimer's disease, and may be the subject before they were administered Alzheimer's disease medication.

[0080] Steps (S41) to (S43) and steps (S41a) to (S43a) can be carried out by performing the same operations as steps (S31) to (S33) of the method of the third embodiment. However, in steps (S41) to (S43), a sample derived from the subject is used, and in steps (S41a) to (S43a), a sample derived from the control is used. In the method for diagnosing Alzheimer's disease, steps (S41a) to (S43a) can be executed in parallel with steps (S41) to (S43).

[0081] The present inventors have revealed that the visualization agent and, consequently, the diagnostic agent of the first embodiment (particularly CAP-C-PF-alkyne) preferentially labels the initial aggregated state of amyloid-beta protein and fibrilized Tau441(2N4R)P301S (as will be explained in the experimental examples below). If the diagnostic agent has a click reaction site, the presence or absence of labeled protein can be determined by comparing the results of mass spectrometry in step (S44). When performing mass spectrometry in step (S44), it is preferable to add the click reaction reagent to the target mixture and the control mixture after steps (S41) and (S41a) to introduce functional groups into the labeled protein, and then perform a concentration operation corresponding to the functional group (for example, reacting with a biotinylation reagent and adding avidin beads). By comparing the results of mass spectrometry, the presence or absence of amyloid-beta protein and Tau441(2N4R)P301S can be determined, and consequently, the presence or absence of Alzheimer's disease can be determined. Mass spectrometry allows for comprehensive analysis of each protein. Furthermore, applying mass spectrometry is preferable because it allows the binding affinity of the visualization agent to the target protein to be expressed numerically as label strength and compared with various standards.

[0082] In the method of the fourth embodiment, it was explained that the presence or absence of Alzheimer's disease is determined by comparison in step (S44). However, by appropriately setting the conditions for the subject and the control, it is possible to diagnose conditions other than the presence or absence of Alzheimer's disease. For example, if the control is a past point in time of the subject and it is known that the subject has Alzheimer's disease, it is possible to diagnose the progression of the subject's Alzheimer's disease. Also, if the control is before the drug is administered to the subject who is known to have Alzheimer's disease, it is possible to diagnose the therapeutic effect of the drug on Alzheimer's disease. Furthermore, based on the degree of the therapeutic effect, it can also be used to predict the prognosis of the subject's Alzheimer's disease.

[0083] [Fifth Embodiment] (Method for Diagnosing Parkinson's Disease) The fifth embodiment relates to a method for diagnosing Parkinson's disease, in which the presence or absence of Parkinson's disease in a subject is determined. Each step of this diagnostic method can be performed using the same operations as the corresponding steps (S40) to (S43) and (S41a) to (S43a) of the method of the fourth embodiment.

[0084] Specifically, the diagnostic method of the fifth embodiment includes: (S50) a step of preparing a diagnostic agent that selectively binds to aggregated proteins to form a complex, a sample derived from a subject containing multiple types of proteins, and a sample derived from a control containing multiple types of proteins; (S51) a step of contacting the sample derived from the subject with the visualization agent prepared in step (S50) to obtain a target mixture; (S52) a step of isolating and purifying each protein in the target mixture; (S53) a step of detecting the complex from each protein isolated and purified in step (S52); (S51a) a step of contacting the sample derived from the control with the visualization agent prepared in step (S50) to obtain a control mixture; (S52a) a step of isolating and purifying each protein in the control mixture; (S53a) a step of detecting the complex from each protein isolated and purified in step (S52a); and (S54) a step of comparing the detection results obtained in steps (S53) and (S53a) to determine whether or not the subject has Parkinson's disease.

[0085] The diagnostic agent prepared in step (S50) is the same as the diagnostic agent described as a further embodiment of the first embodiment. That is, the diagnostic agent prepared in step (S50) is the same as the visualization agent described in the first embodiment, the visualization agent prepared in step (S30) of the third embodiment, and furthermore, the visualization agent prepared in step (S40) of the fourth embodiment.

[0086] If the diagnostic agent has a click reaction site, the presence or absence of labeled protein can be determined by comparing the results of mass spectrometry in step (S54). When performing mass spectrometry in step (S54), it is preferable to add the click reaction reagent to the target mixture and the control mixture after steps (S51) and (S51a) to introduce functional groups into the labeled protein, and then perform a concentration operation corresponding to the functional group (for example, reacting with a biotinylation reagent and adding avidin beads). By comparing the mass spectrometry results, the presence or absence of α-synuclein can be determined, and consequently, the presence or absence of Parkinson's disease can be determined.

[0087] In the method of the fifth embodiment, the terms "subject," "control," and "sample" are defined the same as in the method of the fourth embodiment. However, please understand that in the definitions described in the fourth embodiment, Alzheimer's disease should be replaced with Parkinson's disease, and amyloid β1-42 and Tau441(2N4R)P301S should be replaced with α-synuclein.

[0088] [Sixth Embodiment] (Method for identifying proteins that bind to a test compound) The sixth embodiment relates to a method for identifying proteins that bind to a test compound. As shown in Figure 7, the method includes the following steps (1), (A1) to (A4), (B1) to (B4) and (10): (1) A step of preparing the visualization agent of the first embodiment, a sample containing multiple types of proteins, and a test compound; (A1) A step of bringing the sample, test compound, and visualization agent prepared in (1) into contact with each other to obtain a first mixture; (A2) A step of performing a protein denaturation operation on the first mixture after step (A1); (A3) A step of isolating and purifying each protein in the first mixture after step (A2); (A4) A step of subjecting each protein isolated and purified in step (A3) to mass spectrometry; (B1) A step of bringing the sample and visualization agent prepared in step (1) into contact with each other to obtain a second mixture; (B2) A step of performing a protein denaturation operation on the second mixture obtained in step (B1); (B3) A step of isolating and purifying each protein contained in the second mixture obtained in step (B2). (B4) A step of subjecting each protein isolated and purified in step (B3) to mass spectrometry. (10) A step of comparing the analytical results of step (A4) and step (B4), and identifying proteins whose signal intensity obtained in step (A4) is lower than the signal intensity obtained in step (B4) as proteins that bind to the test compound.

[0089] In the method of the sixth embodiment, a sample containing multiple types of proteins, a test compound, and the visualization agent of the first embodiment are prepared (step (1)), and used in each of the subsequent steps. The test compound is not particularly limited as long as it is a compound for which binding to proteins is desired. That is, the test compound may be a ligand for a protein, and may be inorganic or organic.

[0090] In step (A1), a first mixture is obtained by bringing a sample containing multiple types of proteins, a test compound, and a visualization agent into contact with each other. The sample containing multiple types of proteins is not particularly limited and may include, for example, cell lysates or cell extracts. The test compound is also not particularly limited and may include, for example, physiologically active compounds. The contact is not particularly limited, similar to the contact step (S21) of the method in the second embodiment and the contact step (S31) of the method in the third embodiment. For example, a method in which a liquid containing a visualization agent is added to the sample, or a method in which a solid visualization agent is added to the sample, can be used.

[0091] In step (A2), the proteins in the first mixture obtained in step (A1) are subjected to a denaturation operation. The protein denaturation operation may be any of the following: heating, adding a denaturing agent, changing the pH, or applying pressure. For example, the heating conditions in the heating operation are not particularly limited as long as the temperature and time are such that the proteins are denatured. The heating temperature can be, for example, 40 to 80°C, and the heating time can be, for example, 1 to 30 minutes.

[0092] In step (A3), the multiple types of proteins contained in the first mixture obtained in step (A2) are separated and purified into individual proteins. The separation of multiple types of proteins in the sample can be carried out by commonly used methods, such as electrophoresis or gel filtration.

[0093] If the visualization agent has a click reaction site, separation and purification can be carried out by adding a click reaction reagent to the first mixture to introduce a functional group, and then performing a concentration operation corresponding to the functional group. For example, if the functional group is biotin (i.e., the click reaction reagent is a biotinylating reagent), the concentration operation can be carried out by contacting the mixture with avidin beads.

[0094] That is, if the chemical probe molecule of the visualization agent is a compound of formula (I) and R' has a click reaction site, the process may further include a first click reaction step and a second click reaction step. The first click reaction step is to bring a concentration tag molecule bound to the click reaction site of the visualization agent into contact with a protein after step (A1) and before step (A4); and the second click reaction step is to bring a concentration tag molecule bound to the click reaction site of the visualization agent into contact with a protein after step (B1) and before step (B4).

[0095] In the first and second click reaction steps, the first and second mixtures obtained in steps (A1) and (B1), respectively, are brought into contact with the click reaction reagent described in the second embodiment to allow the click reaction to proceed. Since the concentration tag molecule contained in the click reaction reagent can be used as a linker or scaffold molecule, various concentration operations can be performed on the complex of aggregated protein and visualization agent that may be contained in the first and second mixtures, measurements can be performed with higher sensitivity.

[0096] In step (A4), each protein separated in step (A3) is subjected to mass spectrometry. Mass spectrometry can be performed according to conventional methods.

[0097] In step (B1), a sample containing multiple types of proteins is brought into contact with a visualization agent to obtain a second mixture. Step (B1) can be carried out in the same manner as step (A1), except that the test compound is not added.

[0098] In step (B2), the proteins in the second mixture obtained in step (B1) are denatured. Step (B2) can be carried out in the same manner as step (A2).

[0099] In step (B3), several types of proteins contained in the second mixture obtained in step (B2) are separated and purified. Step (B3) can be carried out in the same manner as step (B3).

[0100] In step (B4), each protein separated in step (B3) is subjected to mass spectrometry. Step (B4) can be carried out in the same manner as step (A4).

[0101] Processes (B1) to (B4) may be executed simultaneously with processes (A1) to (A4), or they may be executed at different times.

[0102] In step (10), the signal intensity of each protein measured in step (A4) is compared with the signal intensity of each protein measured in step (B4), and proteins whose signal intensity measured in step (A4) is lower than that measured in step (B4) are determined to be aggregated proteins that bind to the test compound.

[0103] The principle by which proteins that bind to the test compound can be identified by comparing signal intensities is as follows: When the test compound binds to a protein contained in the mixture, that protein acquires resistance to thermal denaturation, and the degree of thermal denaturation decreases. This decrease in the degree of thermal denaturation is detected as a decrease in signal intensity. Therefore, proteins whose signal intensity measured in step (A4) is lower than the signal intensity measured in step (B4) are proteins that have acquired resistance to thermal denaturation and can be determined to be proteins that bind to the test compound.

[0104] The method of the sixth embodiment allows for comprehensive analysis of each protein by using mass spectrometry. Furthermore, using mass spectrometry is preferable because it allows the binding affinity of the visualization agent to the target protein to be expressed numerically as label intensity and compared with various standards.

[0105] [Example] The present invention will be described in more detail below with reference to various experimental data, but the present invention is not limited to these contents.

[0106] Example 1: Screening of chemical probe molecules Fluorescent chemical probe molecules capable of selectively and sensitively identifying aggregated proteins, i.e., usable as visualization agents of the present invention, were screened using the following procedure.

[0107] (Selection of Candidate Compounds) The inventors considered that, as conditions for a fluorescent chemical probe molecule capable of selectively identifying aggregated proteins, it is important that the probe molecule as a whole is hydrophobic, that it is reactive with lysine residues of aggregated proteins, and that the leaving group is appropriately detachable. Furthermore, the inventors considered that, as a condition for high-sensitivity detection, it is important that the chemical probe molecule has a functional group that can introduce a concentration tag molecule for concentrating proteins. As fluorescent compounds that satisfy these conditions, four aminocoumarin analogs (formulas (1) to (4) below) and six N-hydroxysuccinimide (NHS) compounds (formulas (5) to (10) below) were selected. In addition, Octyl-ONBD (formula (11) below), a conventional visualization agent for aggregated proteins, was used as a positive control during screening in the experiment of Example 1.

[0108]

[0109]

[0110] (Binding test to aggregated proteins) Tests were conducted to confirm whether the candidate compounds of formulas (1) to (10) described above could selectively bind to aggregated proteins as chemical probe molecules. First, trastuzumab (20 μM in PBS) was prepared as the protein and contacted with Octyl-ONBD and each of the candidate compounds of formulas (1) to (10). Then, the mixed solution of each candidate compound and trastuzumab was heated (75°C, 15 minutes) to prepare solutions in which the aggregated, heat-denatured trastuzumab was contacted with each candidate compound. Excess low molecular weight compound components were removed from the solutions before and after heating using a Zeba® Spin column (Thermo Fisher Scientific), and the proteins were purified. 5× Laemmli buffer was added to each purified protein solution to a final concentration of 1×. Subsequently, the sample was heated (95°C for 5 minutes), separated by SDS-PAGE, and fluorescence was detected in the resulting gel. Then, CBB staining was performed to confirm the amount of protein contained in the gel.

[0111] (Results) The experimental results for Example 1 are shown in Figures 8 and 9. Referring to Figure 8, it can be seen that the fluorescence intensity of the NHS compounds of formulas (5) to (10) did not change before and after heating, indicating that they did not show selectivity for aggregated proteins. Therefore, it was shown that the NHS compounds of formulas (5) to (10) are unsuitable as visualization agents for aggregated proteins.

[0112] On the other hand, referring to Figure 9, similar to Octyl-ONBD, the fluorescence intensity of all aminocoumarin analogs of formulas (1) to (4) increased from before heating to after heating, indicating selectivity for aggregated proteins. Therefore, it has been shown that aminocoumarin analogs of formulas (1) to (4) can serve as visualization agents for aggregated proteins.

[0113] Example 2: Visualization of Denaturation Resistance by Ligand Addition The experiment in Example 2, described below, measures and verifies the effect of the candidate compounds selected in Example 1 on the denaturation resistance of proteins by ligand addition. The experimental design for Example 2 is explained with reference to Figure 10.

[0114] (Verification Method) Carbonic anhydrase (CA) was prepared as the protein whose denaturation resistance was to be measured. CA was suspended in PBS at a concentration of 5 μM.

[0115] In the experimental setup shown in Figure 10(a), the compounds of formulas (1) to (4), (9), (11), and formulas (12) to (16) below (labeled "probe" in Figure 10(a)) were added as candidate chemical probe molecules.

[0116] In the experimental setup shown in Figure 10(b), first, a final concentration of 10 μM brinzolamide (ligand) was added to the CA solution, and then the compounds of formulas (1) to (4), (9), (11) and formulas (12) to (16) below (referred to as "probe" in Figure 10(b)) were added.

[0117]

[0118] As a positive control, a system with Octyl-ONBD added was also prepared. Measurements were taken according to the individual fluorescence intensity.

[0119] (Results) The measurement results for Octyl-ONBD are shown in Figure 11(a). In Figures 11 to 15, the system without ligand addition is represented by "-", and the system with ligand addition is represented by "+". In the system without ligand addition, almost no fluorescence of the complex was observed below 60°C, but strong fluorescence intensity was observed at 65°C. This indicates that CA was thermally denatured at around 65°C, becoming an aggregated protein, and was able to bind to Octyl-ONBD and emit fluorescence. On the other hand, in the system with ligand addition, almost no fluorescence was observed even at 65°C. From these results, it was confirmed that CA acquires resistance to thermal denaturation by the addition and contact with the ligand, and does not denature or aggregate even when reaching 65°C.

[0120] The measurement results for CAP-C-PF are shown in Figure 11(b). In the system without ligand addition, almost no fluorescence of the complex was observed below 60°C, but strong fluorescence intensity was observed at 65°C. On the other hand, in the system with ligand addition, almost no fluorescence was observed even at 65°C. Therefore, it was confirmed that CAP-C-PF binds selectively to aggregated proteins, similar to the conventional visualization agent Octyl-ONBD. In other words, it was confirmed that CAP-C-PF can be used as a chemical probe molecule for the visualization agent of the present invention.

[0121] The measurement results for CAP-C-Phythal are shown in Figure 12. For comparison of fluorescence intensity, the fluorescence intensity of CAP-C-PF was also measured using the same gel. In the system with ligand added, almost no fluorescence intensity was observed even above 60°C for CAP-C-Phythal, while in the system without ligand added, the fluorescence intensity increased from 60°C to 65°C. However, a small amount of fluorescence intensity was observed even at 60°C, and the fluorescence intensity at 65°C was lower than that observed for CAP-C-PF under the same conditions. Therefore, although CAP-C-Phythal is not optimal considering the observed fluorescence intensity and reaction selectivity, it was confirmed that it has potential to be used as a chemical probe molecule for the visualization agent of the present invention.

[0122] From the results in Figure 12, it was inferred that the reason why the reactivity of CAP-C-Phthal is lower than that of CAP-C-PF is because the leaving group has less leaving ability. Calculating the acid dissociation constants of the conjugate acid of the leaving group, which can serve as an indicator of leaving ability, we found that CAP-C-Phthal is 6.10, CAP-C-Ph is 9.89, and CAP-C-NO ２ 7.15, 7-NET ２ The acid dissociation constants are 6.0 for and 5.5 for CAP-C-PF. In practice, CAP-C-PF and 7-NET showed superior reactivity, as they have relatively small acid dissociation constants. ２ The fact that the compound was CAP-C-Phthal suggests that the acid dissociation constant of the conjugate acid of the leaving group is highly likely to be an indicator of leaving ability, and consequently, selectivity and bonding ability.

[0123] The analogs of CAP-C-PF are CAP-C-2monoF, CAP-C-24diF, CAP-C-26diF, CAP-C-tetraF, CAP-C-Ph, and CAP-C-NO. ２ The measurement results are shown in Figures 13 to 15. For comparison of fluorescence intensity, the fluorescence intensity of CAP-C-PF was also measured using the same gel.

[0124] As shown in Figure 13, no fluorescence was observed for CAP-C-2monoF and CAP-C-24diF at either 60°C or 65°C in the system with ligand added. However, in the system without ligand added, no fluorescence was observed at 60°C, but a very slight fluorescence was observed at 65°C. Therefore, it was shown that CAP-C-2monoF and CAP-C-24diF have lower reactivity to aggregated proteins, i.e., lower desorption properties, compared to CAP-C-PF, and are not excellent as visualization agents for aggregated proteins.

[0125] As shown in Figure 14, CAP-C-26diF did not show fluorescence at either 60°C or 65°C in the ligand-added system, and no fluorescence was observed at 60°C even in the ligand-added system. On the other hand, CAP-C-26diF did show fluorescence at 65°C in the ligand-added system, but its fluorescence intensity was lower than that of CAP-C-PF. In other words, CAP-C-26diF had comparable selectivity for aggregated proteins to CAP-C-PF, but its efficiency was inferior, and it did not detect aggregated proteins with high sensitivity. On the other hand, CAP-C-tetraF showed almost the same changes as CAP-C-PF under the conditions of ligand addition or absence and the combination of 60°C or 65°C. In other words, CAP-C-tetraF was shown to be an excellent visualization agent for aggregated proteins.

[0126] As shown in Figure 15, CAP-C-Ph and CAP-C-NO ２ No fluorescence was observed at 60°C and 65°C in either the system with or without the ligand.

[0127] The acid dissociation constants for each conjugate acid are 5.7 for CAP-C-tetraF and CAP-C-NO ２ The values ​​were 7.15 for CAP-C-24diF and CAP-C-26diF, 7.3 for CAP-C-2monoF, 8.7 for CAP-C-Ph, and 9.89 for CAP-C-Ph. Therefore, it was suggested that the acid dissociation constant of the chemical probe molecule is preferably 6.0 or less, and particularly preferably between 5.5 and 5.7.

[0128] The measurement results for CAP-C-PF-alkyne are shown in Figure 16. No fluorescence was observed for CAP-C-PF-alkyne at either 60°C or 65°C in the system with the ligand added, and no fluorescence was observed at 60°C even in the system with the ligand added. On the other hand, fluorescence was observed for CAP-C-PF-alkyne at 65°C in the system with the ligand added, but its fluorescence intensity was lower than that of CAP-C-PF. This indicates that CAP-C-PF-alkyne has slightly lower reactivity to aggregated proteins than CAP-C-PF, but its selectivity is equivalent. Therefore, it was confirmed that CAP-C-PF-alkyne can be used as a chemical probe molecule for the visualization agent of the present invention.

[0129] Example 3: Comparison of reactivity through competitive experiments with Octyl-ONBD

[0130] In Example 3, the difference in reaction efficiency between Octyl-ONBD and CAP-C-PF was investigated by measuring the effect of adding CAP-C-PF as a competitive probe on fluorescence intensity during the fluorescent labeling of aggregated proteins with Octyl-ONBD. The experimental design for Example 3 is explained with reference to Figure 17.

[0131] (Verification Method) Carbonic anhydrase (CA) was prepared as the aggregated protein. CA was suspended in PBS at a concentration of 5 μM.

[0132] Figure 17(a) shows an experiment in which aggregated CA was labeled with Octyl-ONBD by adding Octyl-ONBD to a final concentration of 10 μM or 100 μM and heating (65°C, 15 minutes). The heated solution was then treated with 5× Laemmli buffer to a final concentration of 1×. Subsequently, the solution was heated (95°C, 5 minutes), the heated sample was separated by SDS-PAGE, and fluorescence at the fluorescence wavelength derived from the N-NBD structure produced by the reaction of Octyl-ONBD was detected in the resulting gel. CBB staining was then performed to confirm the amount of protein contained in the gel.

[0133] Figure 17(b) shows an experiment in which aggregated CA was labeled with Octyl-ONBD and CAP-C-PF by simultaneously adding Octyl-ONBD and CAP-C-PF to final concentrations of 10 μM or 100 μM, respectively, and heating (65°C, 15 minutes). After heating, 5× Laemmli buffer was added to the solution to a final concentration of 1×. Subsequently, the solution was heated (95°C, 5 minutes), and the heated sample was separated by SDS-PAGE. Fluorescence at the fluorescence wavelength derived from the N-NBD structure produced by the reaction of Octyl-ONBD was detected in the resulting gel, and then CBB staining was performed to confirm the amount of protein contained in the gel. Note that the binding of CAP-C-PF was not detected at the same fluorescence wavelength as Octyl-ONBD. Therefore, by treating with Octyl-ONBD simultaneously, it is possible to investigate the extent to which the two compounds compete at the same binding site.

[0134] (Results) Figure 18 shows the fluorescence intensity of the complex of Octyl-ONBD and thermally denatured CA. The fluorescence intensity is greater in the system without CAP-C-PF than in the system with CAP-C-PF. An increase in the competitive effect with increasing CAP-C-PF was observed, confirming that a competitive experiment with the addition of CAP-C-PF can be performed under the experimental conditions of Example 3.

[0135] It was confirmed that the fluorescence intensity when 10 μM of CAP-C-PF and 10 μM of Octyl-ONBD were added was lower than the fluorescence intensity when only 10 μM of Octyl-ONBD was added. Furthermore, it was confirmed that the fluorescence intensity when 100 μM of CAP-C-PF and 10 μM of Octyl-ONBD were added was lower than the fluorescence intensity when only 10 μM of CAP-C-PF and 10 μM of Octyl-ONBD were added. Moreover, the fluorescence intensity when 100 μM of CAP-C-PF and 100 μM of Octyl-ONBD were added was about one-tenth of the fluorescence intensity when only 100 μM of Octyl-ONBD was added.

[0136] The experimental results in Example 3 indicate that labeling by Octyl-ONBD is suppressed by competition from CAP-C-PF. Therefore, it can be seen that the labeling efficiency of CAP-C-PF is equivalent to or better than that of Octyl-ONBD.

[0137] Example 4: Identification of ligand-binding proteins in cell lysate. The experiment in Example 4 involved fluorescently labeling aggregated proteins with CAP-C-PF-alkyne and confirming the protein's resistance to denaturation upon ligand addition to verify whether the complex of aggregated proteins and CAP-C-PF-alkyne can be biotin-labeled.

[0138] (Verification Method) HEK293FT cells were disrupted with RIPA buffer (Nacalai Tesque Co., Ltd.) to prepare a protein solution (protein concentration: 3.0 mg / mL). CA was added to this solution to a final concentration of 5 μM to prepare a protein mixture containing CA. To this mixture, 50 μM brinzoamide was added, followed by the addition of CAP-C-PF-alkyne to a final concentration of 1 mM. The mixture was then heated at 65°C for 5 minutes. Subsequently, a click reaction reagent containing biotin molecules was added to bind biotin to the aggregated protein via CAP-C-PF-alkyne. The click reaction was performed by adding 500 μM CuSO4 to the biotinylated mixture. ４ The reaction was carried out under the following conditions: 500 μM BTTAA, 200 μM Biotin-DADPS-azide, and 2.5 mM sodium ascorbate were added, and the mixture was reacted at room temperature for one hour. After the click reaction, the mixture was centrifuged at 15000 x g for 10 minutes at 4°C to separate the pellet from the supernatant. Low molecular weight components were removed from the supernatant using a Zeba Spin (Thermo Fisher Scientific), and the resulting solution was added to the pellet for integration. Protein components were purified from the resulting pellet-solution mixture using a ReadyPrep® 2-D cleanup kit (BioRad).

[0139] For two-dimensional electrophoresis, the long-time protocol of Auto 2D plus (Merck) was followed, and fluorescence images of the resulting gels were obtained. In the comparative experiment, the same experimental procedure was performed without adding brinzoamide (i.e., ligand).

[0140] (Results) The experimental results for Example 4 are shown in Figure 19. Referring to Figure 19, it was confirmed that the complex of aggregated protein and CAP-C-PF-alkyne could be biotinylated. Furthermore, it was confirmed that the labeling of CA changed upon addition of the ligand brinzamide.

[0141] Example 5: Mass Spectrometry of Ligand-Binding Proteins (Verification Method) HEK293FT cells were disrupted with RIPA buffer (Nacalai Tesque Co., Ltd.) to prepare a protein solution (protein concentration: 3.0 mg / mL). CA was added to this solution to a concentration of 5 μM to prepare a protein mixture containing CA. Brinzolamide was added to this mixture to a concentration of 50 μM, followed by the addition of CAP-C-PF-alkyne to a final concentration of 1 mM. The mixture was then heated at 65°C for 5 minutes. Subsequently, a click reaction reagent containing biotin molecules was added to bind biotin to the aggregated proteins via CAP-C-PF-alkyne.

[0142] The click reaction is performed by adding 500 μM CuSO4 to the biotinylated mixture. ４ The reaction was carried out under the following conditions: 500 μM BTTAA, 200 μM Dde Biotin-PEG4-Picolyl azide, and 2.5 mM sodium ascorbate were added, and the mixture was reacted at room temperature for one hour. After the click reaction, the mixture was centrifuged at 15000 x g for 10 minutes at 4°C to separate the pellet from the supernatant. Low molecular weight components were removed from the supernatant using a Zeba Spin (Thermo Fisher Scientific). The pellet was mixed with 50 μL of 1x PTS buffer (100 mM Tris-HCl, pH 8.0, 12 mM Sodium Deoxycholic Acid, 12 mM SLS: Sodium N-Lauroyl Sarcosinate), sonicated for 15 minutes using a sonicator, and then mixed and dissolved using a vortex mixer.

[0143] The dissolved pellet components were treated to remove low-molecular-weight components using Zeba Spin (Thermo Fisher Scientific). The supernatant and dissolved pellet components were combined and diluted with an equal volume of 100 mM Tris-HCl, pH 8.0 buffer. Streptavidin Sepharose ＴＭ 200 μL of High Performance (Cytiva, 17511301, Streptabidin beads) was washed twice with 1 mL of 1x PTS buffer:100 mM Tris-HCl, pH 8.0, buffer (1:4). The washed Streptabidin beads were mixed with the supernatant and pellet components diluted together, and DNAase I was added to a final concentration of 12.5 U / mL and RNAase A to 100 μg / mL. The mixture was reacted in a rotator at 4°C for 2 hours. The mixture was separated by centrifugation (750x g, 4°C, 1 min), and the supernatant was discarded. Subsequently, 1 mL of the following solution was added to suspend the beads, and the mixture was separated by centrifugation (750x g, 4°C, 1 min). The supernatant was collected and discarded, and this process was performed as one washing operation. The beads were washed three times with 1 mL of 1x PTS, and then three times with 1 mL of 20 mM HEPES, pH=7.5, 100 mM NaCl, and 1 M Urea. Furthermore, they were washed three times with 1 mL of 20 mM HEPES, pH=7.5, and 1 M NaCl, replaced with 1 mL of miliQ 1x PTS, and mixed by inversion in a rotor at 4°C for 5 minutes. After centrifugation (750x g, 4°C, 1 min), the supernatant was discarded, and 200 μL of 1x PTS was added. 500 μL of buffer containing 2% hydrazine at a final concentration was added to 1x PTS, lightly voltexed, and then incubated at room temperature for 1 hour.

[0144] The supernatant after bead centrifugation was collected and the volume was reduced by replacing it with 1x PBS using Amicon Ultra-0.5 mL 10K. The resulting concentrated solution was used in the iST kit (PreOmics) desalting cartridge according to the manufacturer's protocol. The obtained peptide solution was measured using nanoLC-MS (Thermo, Orbitrap Fusion), and the obtained data was analyzed using Proteome Discoverer® 3.0 (Thermo).

[0145] (Results) Figure 20 shows fluorescence images of 2DEP from both the ligand-added and ligand-unadded systems, as well as bubble plots of signal intensities detected by mass spectrometry of samples enriched with biotinylated proteins using both the ligand-added and ligand-unadded systems. In the fluorescence images, the area enclosed by the dotted line indicates the complex of CAP-C-PF and aggregated protein, and it was observed that the fluorescence intensity decreased upon ligand addition.

[0146] In the bubble plot of Figure 20, each point represents the mass spectrometry result for each protein. The vertical axis of the bubble plot represents the theoretical molecular weight of each protein, the horizontal axis represents the isoelectric point of each protein, and the size of each point represents the signal intensity obtained by mass spectrometry. In addition, in the bubble plots for the ligand-free and ligand-added systems, the area enclosed by the dotted line indicates the signal intensity of the complex between CAP-C-PF and aggregated protein, specifically the protein corresponding to the known binding protein of the ligand (carbonic ammonium compound II). It can be seen that the addition of the ligand made it more difficult for CAP-C-PF-alkyne to form a complex in order to stabilize it against thermal denaturation.

[0147] Referring to Figure 20, it can be seen that the signal intensity of the complex in the ligand-free system is higher than in the ligand-added system. This signal intensity can be expressed numerically.

[0148] Figure 21 shows the results of a difference analysis of mass spectrometry results for the ligand-added and ligand-unadded systems. Three mass spectrometry measurements were performed for each system, and the values ​​detected for each protein were analyzed using a volcano plot. The horizontal axis of Figure 21 represents log² (ratio of detection intensity of each protein in the ligand-added / ligand-unadded system) in the ratio of the average values ​​of the three measurements. The vertical axis of Figure 20 represents the statistical significance of the signal difference analysis for each protein detected in the three mass spectrometry measurements, and represents -log10 (p-value in statistical testing). Each point in Figure 21 represents an individual protein species, and proteins whose labeling was suppressed by ligand addition are plotted to be located in the upper left of Figure 20. Referring to Figure 21, it can be seen that CA2, the target protein in Example 4, became less likely to be labeled by the ligand, meaning that CA2 exhibits excellent binding affinity to the ligand.

[0149] Example 6: Labeling of amyloid-beta protein in its initial aggregated state The experiment in Example 6, described below, verified the binding affinity of CAP-C-PF-alkyne to amyloid-beta protein in various aggregated states. For comparison, the binding affinity of ThT, a conventional amyloid-beta protein research reagent, and n-Octyl-ONBD, a conventional amyloid-beta protein visualization agent, to amyloid-beta protein in its initial aggregated state was also verified.

[0150] (Verification Method) Amyloid β-protein (human, 1-42) monomer (Peptide Research Institute #4349-v) was dissolved in a 0.05% aqueous ammonia solution and stocked at 230 μM. From the stock solution stored at -80°C, the solution was diluted and mixed to a final concentration of 100 μM Abeta 1-42 (monomer), 20 mM Tris-HCl, pH = 7.4, and 150 mM NaCl, and incubated in a thermo shaker at 600 rpm and 37°C. The solution incubated for 1 hour was treated as the initial agglutination state (Abeta agg.), and the solution incubated for 16 hours was treated as the fibrous state (Abeta fibli).

[0151] For the monomer, initial aggregated state, and fibril state, ThT and CAP-C-PF-alkyne were added at a final concentration of 100 μM. Fluorescence detection was performed every minute using a plate reader (TECAN) heated to 37°C, with a 1 mm amplitude shake between measurements. The fluorescence measurement conditions for ThT were Ex / Em = 485 / 530 nm, and for CAP-C-PF-alkyne, Ex / Em = 360 / 465 nm. The fluorescence intensity at 5 minutes after the start of measurement was used to create the figures.

[0152] In experiments using oligomers, Amyloid beta oligomers (StressMarq #SPR-488) were used. These were diluted and mixed to a final concentration of 100 μM Abeta1-42 (monomer), 20 mM Tris-HCl, pH=7.4, and 150 mM NaCl, to obtain either 100 μM Octyl-ONBD or 100 μM CAP-C-PF-alkyne. Fluorescence detection was performed over time using a plate reader (TECAN) heated to 37°C, with shaking at an amplitude of 1 mm / second. For Octyl-ONBD fluorescence measurement, Ex / Em = 485 / 530 nm was used, and for CAP-C-PF-alkyne, Ex / Em = 360 / 465 nm was used.

[0153] (Results) Figure 22(a) shows the relative fluorescence intensities observed for ThT and CAP-C-PF-alkyne against amyloid-beta protein in various aggregated states. It can be seen that CAP-C-PF-alkyne showed superior binding affinity to ThT in the initial aggregated state of amyloid-beta protein.

[0154] Figure 22(b) shows the relative fluorescence intensities observed for n-Octyl-ONBD and CAP-C-PF-alkyne, respectively, against amyloid-beta protein oligomers. It can be seen that CAP-C-PF-alkyne showed superior binding affinity to n-Octyl-ONBD in the initial aggregation state of amyloid-beta protein.

[0155] Example 7: Additional fluorescent compounds were selected as candidate probes and their suitability as probes was verified.

[0156] (Verification method) Further candidate fluorescent compounds are six aminocoumarin analogs shown in formulas (17) to (22) below.

[0157] The verification method for Example 7 is the same as the binding test for Example 1. Specifically, trastuzumab (20 μM in PBS) was prepared as the protein and brought into contact with each of the candidate compounds of formulas (17) to (22) above to prepare a mixed solution. Hereinafter, the candidate compound of formula (17) will be referred to as "CAP-C-BzOxZ". Formula (18) will be referred to as "CAP-C-BzThZ", formula (19) as "CAP-C-345triF", formula (20) as "CAP-C-234triF", formula (21) as "CAP-C-246triF", and formula (22) as "CAP-C-HyPyr". Furthermore, as control systems, CAP-C-PF, CAP-C-24diF, CAP-C-26diF, and CAP-C-tetraF were also prepared, and mixed solutions with carbonic anhydrase (CA) were prepared for each.

[0158] In the mixed solution, CA was suspended in PBS at a concentration of 5 μM, and the candidate compound was suspended at a concentration of 100 μM. Each of the prepared mixed solutions was heated for 5 minutes. Multiple temperature conditions were set for heating, and multiple samples were prepared for the candidate compound and control system at different heating temperatures. After heating, 5× Laemmli buffer was added to the solution to a final concentration of 1×. Subsequently, the solution was heated (95°C, 5 minutes), and the heated sample was separated by SDS-PAGE. Fluorescence at the fluorescence wavelength produced by the reaction of the candidate compound was detected in the resulting gel, and then CBB staining was performed to confirm the amount of protein contained in the gel. For CAP-C-345triF, CAP-C-234triF, and some control systems, the experiment was performed twice under the same conditions to confirm reproducibility.

[0159] (Results) The experimental results for Example 7 are shown in Figures 23 to 25.

[0160] Figure 23 is a graph showing the results of binding compatibility tests for CAP-C-BzOxZ, CAP-C-BzThZ, and some control systems. Referring to Figure 23(a), it can be seen that CAP-C-BzOxZ shows a clear change in fluorescence intensity at 60°C to 65°C, similar to CAP-C-PF, and can be used as a visualization agent for aggregated proteins. Referring to Figure 23(b), a clear change in fluorescence intensity was also observed for CAP-C-BzThZ, similar to CAP-C-BzOxZ, and therefore it can be used as a visualization agent for aggregated proteins.

[0161] Figure 24 is a graph showing the results of binding tests for CAP-C-345triF, CAP-C-234triF, and some control systems, with (b) being the result of a replication experiment of (a). Referring to (a) and (b) in Figure 24, almost no change in fluorescence intensity was observed with CAP-C-345triF. That is, it can be understood that CAP-C-345triF shows almost no binding affinity to aggregated proteins. On the other hand, CAP-C-234triF showed a slight change in fluorescence intensity in the temperature range from 60°C to 65°C. Therefore, it was shown that CAP-C-234triF can be used as a visualization agent for aggregated proteins.

[0162] Figure 25 is a graph showing the results of binding tests for CAP-C-246triF, CAP-C-HyPyr, and some control systems. Referring to Figure 25, a slight change in fluorescence intensity was observed for CAP-C-HyPyr between 60°C and 65°C. On the other hand, the change in fluorescence intensity of CAP-C-246triF between 60°C and 65°C was more pronounced than that of CAP-C-HyPyr, CAP-C-345triF, and CAP-C-234triF. Therefore, it can be concluded that CAP-C-HyPyr and CAP-C-234triF can be used as visualization agents for aggregated proteins.

[0163] Based on the experimental results of Examples 1 to 7 above, it can be understood that the presence of a phenyl group with a fluorine atom substituted at the ortho position in the aminocoumarin analog of formula (I) is important for further demonstrating its effectiveness as a visualization agent for aggregated proteins.

[0164] Example 8: Labeling of other protein aggregate nuclei. Example 8 is an experiment aimed at verifying the binding affinity of CAP-C-PF-alkyne to proteins that form aggregate nuclei other than amyloid-beta protein. In Example 8, as a control, the binding affinity of n-Octyl-ONBD, a conventional visualization agent for aggregate proteins, to aggregate nuclei other than amyloid-beta protein was also verified.

[0165] (Verification Method) Tau441(2N4R)P301S and α-synuclein were used in the experiment as proteins that form aggregate nuclei. Amyloid β1-42, whose binding affinity was confirmed in Example 6, was also used as a positive control. Specifically, different aggregate states (monomer state and fibril state or oligomer (aggregate nucleus) state) were prepared for amyloid β1-42 monomer, Tau441(2N4R)P301S, and α-synuclein.

[0166] For each aggregated protein, CAP-C-PF-alkyne or n-Octyl-ONBD was added at a final concentration of 100 μM to bring them into contact and prepare a mixed solution. The mixed solution was subjected to fluorescence detection. Fluorescence detection was performed every minute using a plate reader (TECAN) heated to 37°C, with shaking of 1 mm per second in between.

[0167] (Results) Figure 26 is a graph showing the experimental results of Example 8, where the vertical axis shows the observed fluorescence intensity and the horizontal axis shows the reaction time after adding CAP-C-PF-alkyne to each protein. As shown in Figure 26, Tau441(2N4R)P301S and α-synuclein, like amyloid β1-42, show a clear difference in the fluorescence intensity observed between the monomer state and the fibril state. In other words, CAP-C-PF-alkyne was shown to have excellent binding affinity to Tau441(2N4R)P301S and α-synuclein in the fibril state (i.e., the initial aggregation state).

[0168] Tau441(2N4R)P301S is known as a biomarker for Alzheimer's disease. CAP-C-PF-alkyne, which can selectively identify the initial aggregation state of amyloid β1-42, is considered an excellent diagnostic agent for Alzheimer's disease. Furthermore, since α-synuclein, the initial aggregation state, is known as a biomarker for Parkinson's disease, CAP-C-PF-alkyne can also be used as a diagnostic agent for Parkinson's disease.

[0169] Example 9: Detection of synuclein condensation nuclei by spike-in experiment. Example 8 demonstrated that CAP-C-PF-alkyne selectively binds to α-synuclein in the condensation nucleus state rather than in the monomer state. Example 9 is an experiment to reinforce the results of Example 8. Specifically, it was confirmed whether detection is possible even if a known method for detecting synuclein condensation nuclei is modified to use CAP-C-PF-alkyne.

[0170] (Detection Method) As a known method for detecting synuclein agglutination nuclei, we prepared the commercially available Human Alpha Synuclein Sandwich ELISA Kit (manufactured by Proteintech Group, Inc.). This kit specifically captures α-synuclein with a capture antibody (specifically, an anti-synuclein antibody) on a plate, and then specifically binds α-synuclein to a detection antibody, which is a conjugate of a fluorescent substance, thereby enabling fluorescence detection of α-synuclein.

[0171] In Example 9, the detection antibody in the kit was replaced with a CAP-C-PF-alkyne biotin conjugate and used for fluorescence detection of α-synuclein. The amount of CAP-C-PF-alkyne bound was quantified by introducing biotin using a click reaction and then measuring the emission intensity of the chemiluminescence reaction of Streptavidin-HRP. Three types of samples were measured: plasma samples collected from healthy individuals (hereinafter referred to as "healthy individual samples"), samples of healthy individual plasma to which 6 pmol / L of α-synuclein was added (hereinafter referred to as "standard additive samples"), and samples of healthy individual plasma to which 600 pmol / L of α-synuclein was added (hereinafter referred to as "spike-in samples").

[0172] (Results) Figure 27 is a graph showing the measurement results for Example 9, where (a) shows the luminescence intensity detected for a healthy sample, (b) for a standard-added sample, and (c) for a spike-in sample. Referring to Figure 27, a significant increase in detection signal intensity was confirmed, corresponding to the addition of α-synuclein standard and spike-in. Therefore, since CAP-C-PF-alkyne can replace detection antibodies in existing detection kits, it can be understood that it has sufficient performance as a visualization agent and diagnostic agent for α-synuclein.

[0173] Example 10: Binding site analysis between probe molecule and synuclein. Example 10 relates to binding analysis aimed at explaining the binding structure between the probe molecule and synuclein.

[0174] (Analysis Method) The probe molecule used was CAP-C-PF-alkyne. Preformed fibril (Type 1) of α-synuclein (StressMarq Biosciences Inc., SPR-322C) (10 μM in PBS) was prepared and contacted with CAP-C-PF-alkyne (100 μM) to prepare a mixed solution. After reacting at 25°C for 20 minutes, excess probe was removed using a Zeba Spin column (Thermo Fisher Scientific) to purify the protein. Mass spectrometry samples were prepared from each purified protein solution using an iST kit (PreOmics) according to the manufacturer's protocol. The obtained samples were measured using nanoLC-MS (Orbitrap Fusion®, manufactured by Thermo Fisher Scientific), and the obtained data was analyzed using Proteome Discoverer 3.0 (manufactured by Thermo Fisher Scientific).

[0175] (Results) Mass spectrometry revealed that the theoretical and experimental values ​​of the peptide fragmentation pattern predicted when CAP-C-PF-alkyne was bound to each Lys residue matched. This indicates that CAP-C-PF-alkyne selectively modifies the K45 and K58 domains of α-synuclein, using them as the main labeling sites, meaning that the binding sites for the synuclein probe molecule are K45 and K58.

[0176] The above analysis revealed the binding mode between CAP-C-PF-alkyne and α-synuclein. Previous studies have reported PET probes that exhibit selective binding to synuclein aggregates in the body, and their binding modes have also been clarified. For example, PET probe F0502B has been reported to bind in a manner that modifies the K80 domain of α-synuclein. By referring to these previous studies and the findings obtained in Example 10, it is possible to design probes in a similar chemical space. Furthermore, by obtaining multiple types of probe molecules and antibodies against the probe binding sites, structural identification of aggregation polymorphisms can be detected using a simple method. As described above, based on the contents of this embodiment and examples, a diagnostic method that provides the new value of stratification of synucleinopathy can be provided.

[0177] The embodiments are illustrative, and the scope of the invention is not limited thereto. Various omissions, substitutions, and modifications can be made without departing from the spirit of the invention.

Claims

1. A visualization agent for the aggregation state of proteins, comprising a chemical probe molecule that binds to an aggregated protein to form a complex, wherein the chemical probe molecule is an aminocoumarin analog having a leaving group that is released upon the formation of the complex.

2. The visualization agent according to claim 1, wherein the aminocoumarin analog has a coumarin skeleton containing a tertiary amino group as a substituent.

3. The aminocoumarin analogs are given by the following formula (I) (In the formula, R and R' are the group consisting of a chain hydrocarbon group having 1 to 12 carbon atoms, a branched hydrocarbon group having 3 to 10 carbon atoms, an alicyclic hydrocarbon group having 3 to 10 carbon atoms, and an aryl group having 3 to 10 carbon atoms (however, one or more -CH groups in the chain hydrocarbon group) ２ -CH ２ - is replaced by -CO-NH- or -CO-O-, or one or more -CH in the chain hydrocarbon group ２ -CH ２ -CH ２ The visualization agent according to claim 2, wherein the - is substituted with -NH-CO-NH- or -O-CO-NH-, or one hydrocarbon group independently selected from the branched hydrocarbon group or the alicyclic hydrocarbon group, in which one carbon atom or two or more non-adjacent carbon atoms are substituted with a nitrogen atom or an oxygen atom, and X is represented as a phenyl group, benzoxazole ring, benzothiazole ring, or pyrimidine ring with the ortho position substituted with a fluorine atom, and the X is the leaving group.

4. The visualization agent according to claim 3, wherein the phenyl group whose ortho position is substituted with a fluorine atom is a trifluorophenoxy group, a tetrafluorophenoxy group, or a pentafluorophenoxy group.

5. The visualization agent according to claim 3, wherein R and / or R' is the chain-type hydrocarbon group and is a saturated hydrocarbon group.

6. The visualization agent according to claim 3, wherein the aryl group is a benzyl group or a phenyl group.

7. The visualization agent according to claim 3, wherein the chain-like hydrocarbon group has 2 to 8 carbon atoms.

8. The visualization agent according to claim 3, wherein the branched hydrocarbon group and the alicyclic hydrocarbon group have 3 to 8 carbon atoms.

9. The visualization agent according to claim 3, wherein the pKa of the conjugate acid of X is 6.0 or less.

10. The visualization agent according to claim 3, wherein the pKa of the conjugate acid of X is 5.5 or more and 5.7 or less.

11. The visualization agent according to claim 3, wherein R' has a click reaction site, and the click reaction site has a carbon-carbon triple bond, or is an azide group, a tetrazine structure, a cyclooctin structure, or a cyclooctene structure.

12. A method for visualizing the aggregation state of a protein, comprising: a contact step of bringing an aggregated protein into contact with any one of claims 1 to 10 of a visualizing agent that binds to the aggregated protein to form a complex; and a detection step of detecting the fluorescence of the complex.

13. A method for detecting the presence or absence of aggregated proteins in a sample containing multiple types of proteins, comprising: (S30) preparing a sample containing multiple types of proteins including aggregated proteins, and one visualization agent from claims 1 to 10 that binds to the aggregated proteins to form a complex; (S31) contacting the sample with the visualization agent to obtain a mixture; (S32) isolating and purifying each protein in the mixture; and (S33) detecting the complex from each of the isolated and purified proteins.

14. The method according to claim 13, wherein the visualization agent used in step (S31) is the visualization agent of claim 10, and further comprises a click reaction step of reacting the complex with a concentration tag molecule bound to the click reaction site after step (S31) and before step (S33).

15. A method for diagnosing Alzheimer's disease, comprising: (S40) a step of preparing a visualization agent that selectively binds to aggregated proteins to form a complex, a sample derived from a test subject containing multiple types of proteins, and a sample derived from a control containing multiple types of proteins; (S41) a step of contacting the sample derived from the test subject with the visualization agent prepared in step (S40) to obtain a target mixture; (S42) a step of isolating and purifying each protein in the target mixture; (S43) a step of detecting the complex from each protein isolated and purified in step (S42); (S41a) a step of contacting the sample derived from the control with the visualization agent prepared in step (S40) to obtain a control mixture; (S42a) a step of isolating and purifying each protein in the control mixture; (S43a) a step of detecting the complex from each protein isolated and purified in step (S42a); and (S44) A method comprising the step of comparing the detection results obtained in step (S43) and step (S43a) to determine whether or not the subject has Alzheimer's disease.

16. A method for diagnosing Parkinson's disease, comprising: (S50) a step of preparing a visualization agent that selectively binds to aggregated proteins to form a complex, a sample derived from a test subject containing multiple types of proteins, and a sample derived from a control containing multiple types of proteins; (S51) a step of contacting the sample derived from the test subject with the visualization agent prepared in step (S50) to obtain a target mixture; (S52) a step of isolating and purifying each protein in the target mixture; (S53) a step of detecting the complex from each protein isolated and purified in step (S52); (S51a) a step of contacting the sample derived from the control with the visualization agent prepared in step (S50) to obtain a control mixture; (S52a) a step of isolating and purifying each protein in the control mixture; (S53a) a step of detecting the complex from each protein isolated and purified in step (S52a); and (S54) A method comprising the step of comparing the detection results obtained in step (S53) and step (S53a) to determine whether or not the subject has Parkinson's disease.

17. A method for identifying aggregated proteins that bind to a test compound, comprising the following steps: (1) preparing a sample containing multiple types of proteins, a test compound, and one visualization agent according to any one of claims 1 to 10 that binds to aggregated proteins to form a complex; (A1) contacting the sample prepared in step (1) with the test compound and the visualization agent to obtain a first mixture; (A2) after step (A1), subjecting the first mixture to a protein denaturation operation; (A3) after step (A2), isolating and purifying each protein in the first mixture; (A4) subjecting each protein isolated and purified in step (A3) to mass spectrometry; (B1) contacting the sample prepared in step (1) with the visualization agent prepared in step (1) to obtain a second mixture; (B2) subjecting the second mixture obtained in step (B1) to a protein denaturation operation. A method comprising: (B3) isolating and purifying each protein contained in the second mixture obtained in step (B2); (B4) subjecting each of the proteins isolated and purified in step (B3) to mass spectrometry; and (10) comparing the analytical results obtained in step (5) and step (9), and identifying the proteins whose signal intensity obtained in step (5) is lower than the signal intensity obtained in step (9) as aggregated proteins that bind to the test compound.

18. The method according to claim 17, wherein the visualization agent prepared in step (1) is the visualization agent of claim 10, and further comprises: a first click reaction step of contacting the protein with a concentration tag molecule bound to the click reaction site of the visualization agent after step (A1) and before step (A4); and a second click reaction step of contacting the protein with a concentration tag molecule bound to the click reaction site of the visualization agent after step (B1) and before step (B4).

19. The following formula (I) (In the formula, R and R' are each independently selected from the group consisting of a chain hydrocarbon group having 1 to 12 carbon atoms, a branched hydrocarbon group having 3 to 10 carbon atoms, an alicyclic hydrocarbon group having 3 to 10 carbon atoms, and an aryl group having 3 to 10 carbon atoms (however, one or more -CH ２ -CH ２ - in the chain hydrocarbon group is substituted with -CO-NH- or -CO-O-, or one or more -CH ２ -CH ２ -CH ２ - in the chain hydrocarbon group is substituted with -NH-CO-NH- or -O-CO-NH-, or one carbon atom or two or more non-adjacent carbon atoms in the branched hydrocarbon group or the alicyclic hydrocarbon group may be substituted with a nitrogen atom or an oxygen atom)), and X is a phenyl group in which the ortho position is substituted with a fluorine atom, a benzoxazole ring, a benzothiazole ring, or a pyrimidine ring), an aminocoumarin analog represented by

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